TWI221482B

TWI221482B - Osteoprotegerin

Info

Publication number: TWI221482B
Application number: TW86104638A
Authority: TW
Inventors: William J Boyle; David L Lacey; Frank J Calzone; Ming-Shi Chang
Original assignee: Amgen Inc
Priority date: 1996-09-03
Filing date: 1997-04-11
Publication date: 2004-10-01
Also published as: ID17460A

Abstract

The present invention discloses a novel secreted polypeptide, termed osteoprotegerin, which is a member of the tumor necrosis factor receptor superfamily and is involved in the regulation of bon6 metabolism. Also disclosed are nucleic acids encoding osteoprotegerin, polypeptides, recombinant vectors and host cells for expression, antibodies which bind OPG, and pharmaceutical compositions. The polypeptides are used to treat bone diseases characterized by increased resorption such as osteoporosis.

Description

1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（1 ) 本發明係概括地有關參與骨新陳代謝的調節之多肽。更特疋τ之，本發明係有關一種新穎的多肽，稱爲促骨堆積肤’其爲腫瘤壞死因子受體超族的一成員。該多肽可用來治療具有增加骨耗損特徵的骨疾病例如骨稀鬆病。碧:明背景多肽生長因子和細胞激素皆爲分泌出來的因子，其經由特定地結合到個別，表面結合受體而發出有關廣多種細胞生長’分化’和新陳代謝的變化之信號。以其爲一蛋白質類別’因此受體就共構造和信號傳導方式而言互有不同。彼等的特徵在於其具有一細胞外域以參與配體結合，及細胞質域以傳遞恰當的細胞内信號。受體表現樣式最後決定那些細胞會對應所給配體，而所給受體的構造則指定經由配體結合所謗發的細胞回應。受體等經證明可透過其細胞質域經由活化蛋白質酪胺酸，或蛋白質絲胺酸/蘇胺酸磷酸化 (如，血小板衍生生長因子受體（PDGFR)或轉形生長因子-/? 受體-I(TGFy3R-I))，經由刺激G-蛋白質活化（例如，卢-腎上腺素功能受體），及經由調制與細胞質信號轉導蛋白質的缔合（如，TNFR-1和Fas/APO)而傳遞細胞内信號（Heldin， Cell 脸，213-223(1995)) 〇腫瘤壞死因子受體（TNFR)超族爲一組第I類透膜蛋白質，彼等共有守恒的富含半胱胺酸圖式（motif)，其在細胞外域中重複三至六次（Smith，et al. Cell 76, 953-862(1994))。整體地，這些重複單位形成這些受體的配體結合域（Chen et al.， -4- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） I---------— (請先閲讀背面之注意事項再填寫本頁)1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the Invention (1) The present invention is generally related to polypeptides involved in the regulation of bone metabolism. More specifically, the present invention relates to a novel polypeptide called osteoproliferative peptide, which is a member of the tumor necrosis factor receptor superfamily. The polypeptide can be used to treat bone diseases such as osteoporosis, which are characterized by increased bone loss. Bi: Bright background Both peptide growth factors and cytokines are secreted factors. They specifically bind to individual, surface-bound receptors and send signals about a wide variety of cell growth 'differentiation' and metabolism changes. It is a protein class' so the receptors differ from each other in terms of co-construction and signaling. They are characterized by having an extracellular domain to participate in ligand binding, and a cytoplasmic domain to transmit appropriate intracellular signals. Receptor expression patterns ultimately determine which cells will correspond to a given ligand, and the structure of a given receptor will designate a cellular response via ligand binding. Receptors, etc., have been shown to pass through their cytoplasmic domain via activated protein tyrosine, or protein serine / threonine phosphorylation (eg, platelet-derived growth factor receptor (PDGFR) or transforming growth factor- /? Receptors) -I (TGFy3R-I)), via stimulation of G-protein activation (eg, Lu-adrenergic receptors), and modulation of association with cytoplasmic signal transduction proteins (eg, TNFR-1 and Fas / APO) It transmits intracellular signals (Heldin, Cell face, 213-223 (1995)). The tumor necrosis factor receptor (TNFR) superfamily is a group of type I transmembrane proteins. They share a conserved cysteine-rich A motif, which is repeated three to six times in the extracellular domain (Smith, et al. Cell 76, 953-862 (1994)). Overall, these repeating units form the ligand-binding domains of these receptors (Chen et al., -4- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) I ------- --- (Please read the notes on the back before filling this page)

、1T 1221482 經濟部中央標準局員工消費合作社印製 A7 B7五、發明説明（2 ) Chemistry 270? 2874-2878(1995))。這些受體的配體爲一組與 TNF汉同系，在構造上相關的蛋白質（Goeddel et al· Cold Spring Harbor Symp. Quart. Biol. 5i, 597-609(1986) ； Nagata et al. Science 267, 1449-1456(1995))。TNF泛係結合到個別但彼此密切關聯的受體，TNFR-1和TNFR-2。TNF仪會在載著受體的細胞内產生多種生物回應，包括，增生，分化及細胞毒性和 apoptosis(Beutler et al. Ann. Rev. Biochem. 57., 505-518(1988))。 TNFα被認爲會媒介成急性和慢性炎性回應（Beutler et al. Ann. Rev. Biochem· il，505-508(1988))。TNF從的系統性輸送令謗發毒性休克和遍佈的組織壞死。因此之故，TNFa可能促成與多和感染性疾病，包括敗血病相關的嚴重罹病率和死亡率。FasL，TNFR-相關受體Fas/APO的配體（Suda et al. Cell Zi，1169-1178(1993))，其突變係與自體免疫相關者 (Fisher et al. Cell 紅，935-946(1995))，而 FasL的過度產生可能涉及藥物謗導的肝炎。因此，各種TNFR-相關性蛋白質的配體常媒介成許多疾病狀態的嚴重效應，據此可推測可中和這些配體的活性之藥劑皆具有治療價値。可溶性TNFR-1 受體，及結合TNF々的抗體皆經檢驗過中和系統性TNF從之能力（Loetscher et al· Cancer Cells 3(6)· 221-226(1991))。分泌 TNFR-1 mRNA的自然發生形式已在最近被選殖，且其產生也經檢測過其在活體外（in vitro)和活體内（in vivo)中和TNF從活性之能力（Kohno et al· PNAS USA α，8331-8335(1990))。此蛋白質中和TNF α的能力可推測可溶性TNF受體具有結合 (請先閱讀背面之注意事項再填寫本頁) -5- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（3 ) 和清除TNF的功能藉而阻斷對載有TNFR的細胞所施加之細胞毒性效應。本發明的一項目的爲鑑定出TNFR超族的新成員。經預期該新族員可爲透膜蛋白質或其可溶形式，其包括細胞外域而缺乏透膜和細胞質域。我像鑑定出一新的TNFR超族成員，其編碼一與TNFR-2密切相關的分泌蛋白質。類似於可溶性TNFR-1者，該TNFR-2相關性蛋白質可負面地調節其配體的活性，因而可用來治療某些人類疾病。發明概述一種新穎的腫瘤壞死因子受體（TNFR)超族成員已從胎鼠腸内cDNA庫鑑別出來。得到全長度的cDNA株並予以定序。該老鼠cDNA在導入外來基因的小鼠體内之表現揭露出骨密度有顯著的增加，特別是在長骨，骨盆骨和椎骨之中。該 cDNA編碼的多肽稱爲促骨堆積肽（OPG)且其在促進骨蓄積中具有主要作用地位。經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 本發明提出核酸，其編碼具有至少一種OPG所具生物活性之多肽。此外也提出可雜合到編碼小鼠，老鼠或人類OPG的核酸之核酸，如圖 2B-2C(SEQ ID NO : 120)，9A-9B(SEQ ID NO : 122)，和 9C_9D(SEQ ID NO : 124)中所示者。較佳者，該OPG爲哺乳動物OPG且更佳者爲人類OPG。此外也包括表現OPG的重組媒體和宿主細胞以及製備重組OPG之方法。另外也揭示可特定地結合該多肽的抗體或其片段。本發明也提出治療骨疾病的方法。該多肽可用來預防骨吸除作用且可用來治療導致骨損失的任何病況，例如骨稀鬆 -6- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 _B7 五、發明説明（4 ) 病，高#5血症，柏哲德氏骨病（Pagefs disease of bone)，及因風濕性關節炎或骨髓炎所致骨損失，及類似者。骨病也可以用本發明核酸經由反意或基因治療法予以治療。本發明更包括含有OPG核酸和多肽的醫學組合物。圖式説明圖1. A.新穎EST LORF的EASTA分析。圖中所示爲與人類 TNFR-2序列排列在一起的推得之FRI_1胺基酸序列。B ·新穎 EST LORF的剖面分析，所示爲與人類TNFR-剖面對排在一起的推得RFI-1胺基酸序列。C. TNFR超族的構造圖，顯示出與新穎FRI-1同系的區域。圖2. 全長度老鼠OPG基因-TNFR超族的一新成員_的構造和序列。Α·ρΜ0Β_Β1·1***體的圖。框格表示LORF在cDNA 序列（粗線）内的位置。黑色框格表信號肽，而灰色橢圓形表示富含半胱胺酸重複序列的位置。B，C.老鼠OPG cDNA 的核酸和蛋白質序列。預測的信號序列係下面畫線部份，而N-聯結糖化潛在部位則以粗體底下劃線字母表出。D，E. OPG與其他 TNFR超族成員 fas(SEQ ID NO : 128) ; tnfrl(SEQ ID NO ： 129) ； sfu-t2(SEQ ID NO ： 130) ； tnfr2(SEQ ID NO ： 131) ; cd40(SEQ ID NO : 132) ; osteo(SEQ ID NO : 133); ngfr(SEQ ID NO ： 134) ； ox40(SEQ ID NO ： 135) ； 41bb(SEQ ID NO : 136)的堆積序列比較（Wisconsin GCG Package， Version 8.1)。圖3· 預測老鼠〇PG蛋白質序列的PepPlot分析（Wisconsin GCG Package，Version 8.1)。Α·老鼠 OPG的略圖表示，顯示本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂 1221482 經濟部中央標準局員工消費合作社印製 A7 五、發明説明（5 ) 出疏水性（上）和親水性（下）胺基酸。也顯示出鹼性（上）和酸性（下）胺基酸。B·卢-片層形成性（上）和0 -片層破壞性（卞）胺基酸殘基，如 Chou and Fasman(Adv· Enz. 47, 45-147(19^8)) 所定義者之顯示。C .々螺旋和/?-片層傾向測量之顯系 (Chou and Fasman，上述）。1.00以上的曲線顯示出α -螺旋和卢-片層結構之傾向。結構可能終止於曲線掉到1.00以下的蛋白質區域。D·沈-形成性（上）或破壞性（下）的殘基之顯示。Ε.類似於典型地出現於沈和卢結構的胺基端之序列的蛋白質序列部份之顯示（Chou and Fasman，同上）。F ·類似於典型地出現於α和卢結構的羧基端之序列的蛋白質序列部份之顯示（Chou and Fasman，同上）。G·典型地出現於回轉處的蛋白質序列部份之顯示（Chou and Fasman，同上）。Η · 序列中各位置處的螺旋疏水性要素之顯示（Eisenberg et al. Proc· Natl· Acad· Sci. USA ϋ，140-144(1984))。I·根據 Kyte and Doolittle(J· Mol. Biol. 157，105-132(1982))和Goldman et al.(評述於 Ann. Rev. Biophys. Biophys· Chem. 15_, 321-353 (1986))的平均 hydrophathy之顯示。圖4 . OPG cDNA在人類組織中的mRNA表現樣式。北方氏吸潰係用32P-標示老鼠cDNA***體（A·左邊兩盤），或人類 cDNA***體（B.右邊盤）作爲探測核酸。圖5 · 在肝細胞内表現OPG cDNA的導入外來基因的小鼠之創造。HE-ΟΡΕ外來基因在小鼠肝中的北方氏吸潰表示。圖6. OPG外來基因導入小鼠的骨密度之增加。盤A-F。對照小鼠。G-J，OPG表現小鼠。在驗屍時，所有動物都經輻 -8 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） c请先閲讀背面之注意事項存填寫本頁)1T 1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of Invention (2) Chemistry 270? 2874-2878 (1995)). The ligands of these receptors are a group of structurally related proteins that are homologous to TNF (Goeddel et al. Cold Spring Harbor Symp. Quart. Biol. 5i, 597-609 (1986); Nagata et al. Science 267, 1449-1456 (1995)). TNF ubiquitin binds to individual but closely related receptors, TNFR-1 and TNFR-2. The TNF meter produces a variety of biological responses in the receptor-carrying cells, including proliferation, differentiation, and cytotoxicity and apoptosis (Beutler et al. Ann. Rev. Biochem. 57., 505-518 (1988)). TNFα is thought to mediate acute and chronic inflammatory responses (Beutler et al. Ann. Rev. Biochem. Il, 505-508 (1988)). Systemic delivery of TNF from toxic shock and widespread tissue necrosis. For this reason, TNFa may contribute to severe morbidity and mortality associated with multiple and infectious diseases, including sepsis. FasL, a ligand of the TNFR-related receptor Fas / APO (Suda et al. Cell Zi, 1169-1178 (1993)), and its mutations are related to autoimmunity (Fisher et al. Cell Red, 935-946 ( 1995)), and the excessive production of FasL may involve drug-induced hepatitis. Therefore, the ligands of various TNFR-related proteins often mediate serious effects in many disease states, and it can be speculated that agents that can neutralize the activity of these ligands have therapeutic value. Soluble TNFR-1 receptors and antibodies that bind TNF々 have been tested for their ability to neutralize systemic TNF (Loetscher et al. Cancer Cells 3 (6) 221-226 (1991)). A naturally occurring form that secretes TNFR-1 mRNA has been recently selected, and its production has also been tested for its ability to neutralize TNF activity in vitro and in vivo (Kohno et al. PNAS USA α, 8331-8335 (1990)). The ability of this protein to neutralize TNF α can be presumed that the soluble TNF receptor has binding (please read the precautions on the back before filling this page) -5- This paper size applies to China National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 5. Description of the invention (3) and the function of eliminating TNF thereby block the cytotoxic effect exerted on the TNFR-loaded cells. It is an object of the present invention to identify new members of the TNFR superfamily. It is expected that this new family member may be a transmembrane protein or a soluble form thereof that includes the extracellular domain and lacks the transmembrane and cytoplasmic domains. I seem to have identified a new member of the TNFR superfamily, which encodes a secreted protein closely related to TNFR-2. Similar to soluble TNFR-1, this TNFR-2 related protein can negatively regulate the activity of its ligand and thus can be used to treat certain human diseases. SUMMARY OF THE INVENTION A novel tumor necrosis factor receptor (TNFR) superfamily member has been identified from the fetal rat intestinal cDNA library. Full-length cDNA strains were obtained and sequenced. The performance of the mouse cDNA in mice introduced with foreign genes revealed a significant increase in bone density, especially in long bones, pelvic bones and vertebrae. The polypeptide encoded by this cDNA is called osteoporosis peptide (OPG) and it has a major role in promoting bone accumulation. Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling this page) The present invention proposes a nucleic acid that encodes at least one polypeptide with biological activity of OPG. Nucleic acids that can be hybridized to nucleic acids encoding mouse, mouse or human OPG are also proposed, as shown in Figures 2B-2C (SEQ ID NO: 120), 9A-9B (SEQ ID NO: 122), and 9C_9D (SEQ ID NO : 124). Preferably, the OPG is a mammalian OPG and more preferably a human OPG. Also included are recombinant media and host cells expressing OPG and methods for making recombinant OPG. Antibodies or fragments thereof that specifically bind to the polypeptide are also disclosed. The invention also proposes a method for treating bone diseases. The peptide can be used to prevent bone resorption and can be used to treat any condition that causes bone loss, such as bone loosener-6- This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) 1221482 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Printed by Consumer Cooperatives A7 _B7 V. Description of the invention (4) Disease, hyper # 5emia, Pagefs disease of bone, and bone loss caused by rheumatoid arthritis or osteomyelitis, and similar By. Bone disease can also be treated with the nucleic acid of the present invention via antisense or gene therapy. The invention further includes medical compositions containing OPG nucleic acids and polypeptides. Schematic illustration Figure 1. A. EASTA analysis of novel EST LORF. The figure shows the deduced FRI_1 amino acid sequence aligned with the human TNFR-2 sequence. B. Profile analysis of the novel EST LORF, showing the RFI-1 amino acid sequence deduced alongside the human TNFR-profile pair. C. Structural map of the TNFR superfamily, showing regions homologous to the novel FRI-1. Figure 2. Construction and sequence of a new member of the full-length mouse OPG gene-TNFR superfamily. Figure of ΑρρΜ0Β_Β1 · 1 insert. The box indicates the position of LORF within the cDNA sequence (thick line). Black boxes indicate signal peptides, while gray ovals indicate positions that are rich in cysteine repeats. B, C. Nucleic acid and protein sequences of mouse OPG cDNA. The predicted signal sequence is underlined, and the potential sites of N-linked saccharification are shown in bold underlined alphabet. D, E. OPG and other TNFR superfamily members fas (SEQ ID NO: 128); tnfrl (SEQ ID NO: 129); sfu-t2 (SEQ ID NO: 130); tnfr2 (SEQ ID NO: 131); cd40 (SEQ ID NO: 132); osteo (SEQ ID NO: 133); ngfr (SEQ ID NO: 134); ox40 (SEQ ID NO: 135); 41bb (SEQ ID NO: 136) stacked sequence comparison (Wisconsin GCG Package, Version 8.1). Figure 3. PepPlot analysis of predicted mouse PG protein sequence (Wisconsin GCG Package, Version 8.1). Α · The mouse OPG sketch shows that this paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the precautions on the back before filling out this page) Order 1221482 Employees' Cooperatives, Central Standards Bureau, Ministry of Economic Affairs Printing A7 5. Description of the invention (5) Hydrophobic (upper) and hydrophilic (lower) amino acids. It also shows basic (top) and acidic (bottom) amino acids. B. Lu-sheet formation (top) and 0-sheet destructive (卞) amino acid residues, as defined by Chou and Fasman (Adv · Enz. 47, 45-147 (19 ^ 8)) Of display. C. Apparent spiral and /?-Lamellar propensity measurements (Chou and Fasman, above). The curve above 1.00 shows the tendency of α-helix and Lu-sheet structure. The structure may end up in a region of the protein where the curve falls below 1.00. D. Shen-Display of forming (top) or destructive (bottom) residues. E. Display of a protein sequence portion similar to the sequence typically found at the amine end of the Shen and Lu structures (Chou and Fasman, supra). F. Display of protein sequence parts similar to sequences typically found at the carboxy terminus of alpha and lu structures (Chou and Fasman, supra). G. Display of the portion of the protein sequence that typically appears at the turn (Chou and Fasman, supra). Η · Display of helical hydrophobic elements at various positions in the sequence (Eisenberg et al. Proc. Natl. Acad. Sci. USA ϋ, 140-144 (1984)). I. According to Kyte and Doolittle (J. Mol. Biol. 157, 105-132 (1982)) and Goldman et al. (Reviewed in Ann. Rev. Biophys. Biophys. Chem. 15_, 321-353 (1986)) The average hydrophathy is shown. Figure 4. mRNA expression pattern of OPG cDNA in human tissues. The Northern blotting system used 32P-labeled mouse cDNA inserts (A. left two plates), or human cDNA inserts (B. right plate) as detection nucleic acids. Figure 5 · Creation of mice expressing OPG cDNA and introducing foreign genes into liver cells. Expression of HE-OPE foreign genes in northern liver suckle in mouse liver. Figure 6. Increased bone density in OPG foreign gene-introduced mice. Plate A-F. Control mice. G-J, OPG express mice. At the time of autopsy, all animals were radiated. -8-This paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) c. Please read the precautions on the back and save this page)

1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（6 ) 一-- 射照像並製備成相片。於A_F中，顯示出諸對照動物和一導卜來基因非表現者（#28)的輻射相片。可注意到該等骨具有清晰明確的表層和透明的中央骨髓腔。相對地，0PG動物 (G-J)具有不明確的表層和增加的骨髓區内密度。圖7· 〇PG外來基因導入小鼠的小梁骨増加情形。A-D對照動物的骨所得代表性顯微照片。於A和B中，顯示出股骨的低（4X，1 〇χ)功率影像（Mass〇n Trichr〇me染色）。抗酒石酸鹽酸性磷酸酶（TRAP)染色顯示出吸收性軟骨（c)和小梁骨 (D)的蝕骨細胞（參看箭頭所示）。注意小梁骨上的蝕骨細胞 <扁平外觀。Ε·Η.取自〇PG-表現動物的骨之代表性顯微照片。於Ε和F中，顯示出股骨的低（4χ，1〇χ)倍率影像 (Masson Trichrome染色）。透明區的生長板軟骨，藍色染色區爲骨，而紅色部位爲骨髓。注意與對照樣相異者，未被吸除的小梁骨導致一般骨髓腔的不存在。此外，所得小梁骨具有藍色和透明部位斑雜的外觀。透明部位爲尚未改型的生長板軟骨之殘餘部份。根據TRAP染色，這些動物在生長板處確實具有蝕骨細胞（參看箭頭，其數目可能減少。不過，遠離生長板的小梁骨表面則幾乎沒有蝕骨細胞 (H)，其爲與對照動物（參看D)有直接相異處之發現。圖8 · HE_OPE表現子在單核細胞-巨噬細胞發展中具有缺陷。小鼠骨質石化病的一個肇因爲M-CSF基因中點突變所致缺陷性M-CSF產生。此爲導致循環系和組織系巨噬細胞的顯著缺乏。由H1E分析評估得知〇pg表現子的周園血液含有單核細胞。爲了確定組織巨嗤細胞的存在，使用可辨識鼠類 -9 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 、1Τ • I - I i^i 1221482 經濟部中央標準局員工消費合作社印製 A7 B7五、發明説明（7 ) 巨噬細胞上的細胞表面抗原之F480抗體進行免疫組織化學分析。A和顯示出得自正常表現子和CR1迥度表現子的脾之低倍率（4X)顯微照片。注意兩種動物都具有許多F480陽性細胞。在正常表現子（B)和HE-ΟΡΕ過度表現子（D)的骨髓中也都含有單核細胞·巨噬細胞（40X)。圖9 · 小鼠和人類OPG cDNA株的構造和序列。A，B。小鼠 cDNA和蛋白質序列。C，D。人類cDNA和蛋白質序列。預測的信號肽係下面畫線者，N-聯結糖化潛在部位則以粗體字表出。E，F。老鼠，小鼠和人類OPG胺基酸序列的序列對正和比較。圖10. TNFR-1和人類OPG細胞外域中的守惺序列之比較。 TNFR1 和 OPG 排列 PrettyPlot(Wisconsin GCG Package, Version 8.1)係在實施例6中説明者。上面行，人類TNFR1序列編碼域1 - 4，底下行，人類OPG序列編碼域1 - 4。守恆殘基係以長方形框格突顯出。圖1 1 ·人類OPG的三次元模型。與人類TNF /?(細線）共結晶的人類OPG殘基25至163(寬線）預測三次元構造之Molescript 顯示的側視圖。作爲方向的參考，沿OPG多肽主鏈的粗箭號係從N_端指向C-端。此外也沿著多肽主鏈插置個別半胱胺酸殘基側鏈的位置以幫助顯現出個別的富半胱胺酸域。TNF 分子係依Banner et al.(1993)所述排列成線者。圖1 2 · OPG畜含半胱胺酸域之構造。將人類（上行，SEQ ID NO : 136)和小鼠（下行）OPG胺基酸序列的比對突顯出預測的 OPG域結構。該多肽係分成兩半部；N_端（A)，和C-端 (請先閲讀背面之注意事項再填寫本頁) 訂 •10- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐）經濟部中央標準局員工消費合作社印製 1221482 A7 B7 五、發明説明（8 ) (B)。N-端半部經預測含有四個富含半胱胺酸域（標爲1-4)。預測的鏈間雙硫鍵係以粗線表出，標爲"SSI”，”SS2n，或 •’SS3n。酪胺酸28和組胺酸75(底下畫線者）爲預期形成離子 ***互作用者。而預期會與OPG配體交互作用的胺基酸係在各恰當殘基上方以粗點表示。位於OPG C-端本部内的半胱胺酸殘基係以長方形框格表出。圖1 3 .全長度與截頭小鼠OPG-Fc融合蛋白質的表現和分泌。A.圖中以箭頭表出融合到人類IgGl Fc域的點。B.得自 Fl.Fc(在白胺酸401融合到Fc的全長度OPG)和CT.Fc(在蘇胺酸180融合到Fc的羧基端截頭OPG)融合蛋白質表現媒體之經調理介質的銀染色結果和SDS-聚丙烯醯胺凝膠分離結果。道1，母pCEP4表現媒體細胞系；道2，Fl.Fc媒體細胞系；道3，CT.Fc媒體細胞系。C.用抗-人類IgGl Fc域爲探測核酸丙從Fl.Fc和CT.Fc蛋白質表現媒體得到的調理介質之西氏方吸潰分析。道1，母pCEG4表現媒體細胞系；道2，Fl.Fc 媒體細胞系；道3，CT.Fc媒體細胞系。1221482 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Description of Invention (6) I-Take a photo and make a photo. In A_F, radiographs of control animals and a leader gene non-expressor (# 28) are shown. Note that the bones have a clear surface layer and a transparent central bone marrow cavity. In contrast, OPG animals (G-J) have an ambiguous surface layer and increased density in the bone marrow region. Fig. 7. Trabecular epiphysis of foreign genes introduced into mice. A-D Representative micrographs taken from animal bones. In A and B, low (4X, 100x) power images of the femur were shown (Masson Trichome staining). Tartrate-resistant acid phosphatase (TRAP) staining showed osteoclasts of resorbable cartilage (c) and trabecular bone (D) (see arrows). Note the osteoclasts on the trabecular bone < flat appearance. E.Η. Representative micrographs taken from OPG-expressing animal bones. In E and F, low (4x, 10x) magnification images of the femur (Masson Trichrome staining) are shown. The growth plate cartilage in the transparent area, bone in the blue stained area, and bone marrow in the red area. Note that in contrast to the control, trabecular bone that has not been aspirated results in the general absence of the bone marrow cavity. In addition, the resulting trabecular bone had a mottled appearance of blue and transparent parts. The transparent area is the remainder of the growth plate cartilage that has not been modified. According to TRAP staining, these animals did have osteoclasts at the growth plate (see arrows, their number may be reduced. However, the surface of the trabecular bone far from the growth plate was almost free of osteoclasts (H), which was comparable to control animals ( (See D) There are direct differences. Figure 8. The HE_OPE epitope has defects in monocyte-macrophage development. A defect in mouse osteopetrosis is caused by a point mutation in the M-CSF gene M-CSF production. This is caused by a significant lack of circulating and tissue line macrophages. H1E analysis revealed that the peripheral blood of the 0pg expressor contained monocytes. To determine the presence of tissue macrophage cells, Identification of rats-9-This paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page), 1T • I-I i ^ i 1221482 Central Standard of the Ministry of Economic Affairs A7 B7 printed by the Bureau ’s Consumer Cooperatives V. Description of the invention (7) Immunohistochemical analysis of F480 antibody against cell surface antigens on macrophages. A and A are shown from the table of normal expressors and CR1 Low magnification (4X) photomicrograph of the spleen of the child. Note that both animals have many F480-positive cells. Monocytes are also contained in the bone marrow of normal expressors (B) and HE-OPE overexpressors (D). • Macrophages (40X). Figure 9 • Structure and sequence of mouse and human OPG cDNA strains. A, B. Mouse cDNA and protein sequences. C, D. Human cDNA and protein sequences. The predicted signal peptide line is below In the line drawing, the potential sites of N-linked saccharification are shown in bold. E, F. Sequence alignment of mouse, mouse and human OPG amino acid sequences. Figure 10. TNFR-1 and human OPG extracellular domain Comparison of the defensive sequences in TNFR1 and OPG. PrettyPlot (Wisconsin GCG Package, Version 8.1) is described in Example 6. Above, human TNFR1 sequence coding domains 1-4, bottom and bottom, human OPG sequence coding domain 1-4. Conserved residues are highlighted in rectangular boxes. Figure 1 1 · Three-dimensional model of human OPG. Human OPG residues co-crystallized with human TNF /? (Thin line) 25 to 163 (wide line) predicted three times Side view of the metastructure of Molescript display. The reference, the thick arrow along the OPG polypeptide main chain is from the N-terminus to the C-terminus. In addition, the positions of the side chains of individual cysteine residues are also inserted along the polypeptide main chain to help show the individual half-rich Cysteine domains. TNF molecules are arranged in a line as described by Banner et al. (1993). Figure 12 · Structure of OPG animal containing cysteine domains. The human (upstream, SEQ ID NO: 136) and An alignment of mouse (bottom) OPG amino acid sequences highlights the predicted OPG domain structure. The peptide is divided into two halves; the N-terminus (A), and the C-terminus (please read the precautions on the back before filling this page). • 10- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 1221482 A7 B7 V. Description of Invention (8) (B). The N-terminal half is predicted to contain four cysteine-rich domains (labeled 1-4). The predicted interchain disulfide bonds are shown in bold lines and are labeled " SSI "," SS2n, or • 'SS3n. Tyrosine 28 and histidine 75 (underlined) are expected to form ionic interactions. Amino acids that are expected to interact with the OPG ligand are shown as thick spots above the appropriate residues. The cysteine residues located in the C-terminal part of the OPG are shown as rectangular frames. Figure 13. Performance and secretion of full-length and truncated mouse OPG-Fc fusion protein. A. The points fused to the human IgGl Fc domain are indicated by arrows in the figure. B. Conditioned media obtained from the expression media of Fl.Fc (full-length OPG fused to Fc at leucine 401) and CT.Fc (truncated OPG fused to carboxyl terminus of threonine 180 to Fc) fusion protein expression media Silver staining results and SDS-polyacrylamide gel separation results. Lane 1, the parent pCEP4 expresses the media cell line; lane 2, the Fl.Fc media cell line; lane 3, the CT.Fc media cell line. C. Wester's square aspiration analysis using the anti-human IgGl Fc domain as a conditioning medium for detection of acyl propionate from Fl.Fc and CT.Fc protein expression media. Lane 1, the parent pCEG4 expresses the media cell line; lane 2, the Fl.Fc media cell line; lane 3, the CT.Fc media cell line.

圖14.人類OPG在大腸样菌（E. coli)中的表現。A.細菌表現媒體的構成。人類OPG基因的LORF經PCR擴大後，接合到寡核甞酸聯結子片段（上面的股爲SEQ ID NO : 137 ;下面的股的SEQ ID NO : 127)，再連接到pAMG21媒體DNA内。所得媒體能夠表現出聯結到N-端甲硫胺酸殘基的OPG殘基32-401。B.載有pAMG21_人類OPG-32-401質體的未經謗導和經謗導細菌之SDS-PAGE分析。道1，MW標準樣；道2，未經謗導的細胞；道3，30X：謗導；道4，37°C謗導；道5，37°C -11 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁) 訂 -钃· 1221482 經濟部中央標準局員工消費合作社印製 A7 __ _B7__ 五、發明説明（9 ) 謗導所得全細胞溶裂物；道6，全細胞溶裂物的可溶性部份；道7，全細胞溶裂物的不溶性部份；道8，得自全細胞溶裂物的經純化包涵體。圖1 5 · CHO細胞内產生的重組鼠類OPG經SDS-PAGE和西方氏吸潰分析之結果。於所示的每一道也加上等量的經CHO 調理介質，且係經用還原性樣品緩衝液（左道），或非還原性樣品緩衝液（右道）處理而製備者。於電泳後，將離析後的蛋白質轉移到耐綸膜，再用抗-OPG抗體予以探測。OPG 的55 kd單體形式和1〇〇 kd二體物形式之相對位置皆以箭頭指示出。圖1 6 .在CHO細胞内產生的重組鼠類OPG之脈衝追踪分析。CHO細胞經35S-甲硫胺酸/半胱胺酸脈衝標記後，追踪一段所示時間。將代謝標記培養物分離到經調理的介質和細胞内，分別製備出清潔劑萃取物，予以澄清’再用抗-OPG抗體予以免疫沈澱。以SDS-PAGE離析免疫沈澱物後，曝光到軟片上。上左和上右兩盤；在非還原條件下分析的樣品。下左和下右兩盤；在還原條件下分析的樣品。上左和下左盤；細胞萃取物。上右和下右盤；經調理培養基萃取物。OPG的55 kd單體形式和100 kd雙體形式之相對流動性係以箭頭示出。圖17.在CTLL-2細胞系中之OPG表現。製備CTLL-2細胞和經CHO-mu OPG[1-401]轉染細胞的無血清經調理培養基，予以濃縮後，以非還原性SDS-PAGE和西方氏吸潰予以分析。左道：CTLL_2經調理培養基。右道：CHO-muOPG經調理培 __ -12- 本紙張尺度適用中國國家檩準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 、τ 1221482 A7 B7 五、發明説明（1〇 ) 養基。OPG的55 kd單體形式和100 kd雙體形式之相對流動性皆以箭頭示出。圖1 8 ·在得自對照小鼠和OPG導入外來基因小鼠的血清樣品和肝萃取物中的OPG表現之偵檢。導入外來基因的小鼠係依實施例4中所述構成的。OPG表現係在SDS-PAGE後用抗-OPG抗體進行西方氏吸潰分析而予以視測。圖19. huOPG[22-401]_Fc融合蛋白質活體外Πη vitro)蝕骨細胞形成之影響。該蚀骨細胞形成檢定係依實施例11A中所述在沒有（對照樣）或有所示量的huOPG[22-401]-Fc融合體之情況中進行的。蚀骨細胞形成係經由對酒石酸鹽酸性鱗酸酶 (TRAP)的組織化學染色而視測的。A. 0PG添加至100毫微克 /毫升。D.OPG添加至0.1毫微克/毫升。E.OPG添加到0.01 毫微克/毫升。F.0PG添加到0.001毫微克/毫升。G.對照樣，未加0PG。圖2 0 .蚀骨細胞培養物TRAP活性隨0PG量增加而減低。於蝕骨細胞形成檢定中加入所示濃度的huOPG[22_401]-Fc融合蛋白質並依實施例11A中所述定量測定TRAP活性。經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁)Figure 14. Performance of human OPG in E. coli. A. Bacterial expression Media composition. LORF OPG gene in humans after PCR amplification, the joined oligonucleotide Chang acid linker fragment (top strand is SEQ ID NO: 137; the following shares SEQ ID NO: 127), and then ligated into pAMG21 media DNA. The resulting media was able to show OPG residues 32-401 linked to the N-terminal methionine residue. B. SDS-PAGE analysis of pAMG21_human OPG-32-401 plastids with and without defamatory and defamatory bacteria. Road 1, MW standard sample; lane 2, cells without slander guide; channel 3,30X: slander guide; abusive guide channels 4,37 ° C; channel 5,37 ° C -11 - This paper applies the Chinese national standard scale (CNS) A4 size (210X 297 mm) (please read the back of the precautions to fill out this page) book - chop · 1,221,482 Ministry of economic Affairs Bureau of standards employees consumer cooperatives printed A7 __ _B7__ V. description of the invention (9) slander The resulting whole cell lysate is obtained; lane 6, the soluble portion of the whole cell lysate; lane 7, the insoluble portion of the whole cell lysate; lane 8, the purified inclusion body obtained from the whole cell lysate. Recombinant murine OPG produced in 1 5 · CHO cells by SDS-PAGE and FIG Western's collapse results absorption analysis. CHO conditioned medium was prepared by, and the system treated with a reducing sample buffer (left lane), or non-reducing sample buffer (right lane) but also in every illustrated plus an equal amount. After electrophoresis, the isolated protein was transferred to a nylon membrane and detected with an anti-OPG antibody. 1〇〇 kd and 55 kd monomeric form relative position of two bodies in the form of OPG was begin arrows indicate. FIG 16. The recombinant murine OPG produced in CHO pulses cell tracking analysis. CHO cells were pulsed with 35S-methionine / cysteine and tracked for the indicated time. The metabolically labeled culture was separated into conditioned media and cells, and detergent extracts were prepared, clarified ', and immunoprecipitated with anti-OPG antibodies. After the immunoprecipitate was isolated by SDS-PAGE, it was exposed to a film. Upper left and upper right plates; samples analyzed under non-reducing conditions. Lower left and lower right two plates; samples analyzed under reducing conditions. Upper left and lower left plate; cell extract. Top right and bottom right plates; extracts from conditioned media. 55 kd monomeric and 100 kd forms of OPG catamaran-based forms of relative mobility shown by arrows. Figure 17. OPG performance in CTLL-2 cell line. Prepared by CTLL-2 cells and CHO-mu OPG [1-401] transfected cells in serum-free conditioned medium was concentrated to give, in a non-reducing SDS-PAGE and Western's absorption collapse be analyzed. Left channel: CTLL_2 conditioned medium. Right Road: CHO-muOPG conditioned training __ -12- This paper applies China national scale quasi-purlin (CNS) A4 size (210X297 mm) (Please read the back of the precautions to fill out this page), τ 1221482 A7 B7 V. Description of the invention (10) Nutrition. 55 kd monomeric and 100 kd forms of OPG form of double opposing arrows show flowability begin. 8. In FIG. 1 Detects and obtained from the control mouse OPG and OPG expression of foreign genes introduced into mouse serum samples and liver extracts of. The mouse line into which the foreign gene was introduced was constructed as described in Example 4. OPG performance was visually inspected by Western blot analysis with anti-OPG antibodies after SDS-PAGE. FIG 19. huOPG [22-401] _Fc Effect of fusion proteins in vitro Πη vitro) Formation of osteoclast cells. This osteoclast formation assay was performed as described in Example 11A without (control) or with the indicated amount of huOPG [22-401] -Fc fusion. Osteogenesis cells were visualized by histochemical staining of tartrate acid squamase (TRAP). A. 0PG was added to 100 ng / ml. D. OPG was added to 0.1 ng / ml. E. OPG was added to 0.01 ng / ml. F. 0PG was added to 0.001 ng / ml. G. In contrast, no 0PG was added. Figure 2. TRAP activity of osteoclast cell culture decreases with increasing amount of OPG. A huOPG [22_401] -Fc fusion protein of the indicated concentration was added to the osteolysed cell formation assay and the TRAP activity was quantitatively determined as described in Example 11A. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the precautions on the back before filling this page)

圖21. 0PG對蝕骨細胞分化終段的影響。huOPG[22-401]_Fc 融合體係在蝕骨細胞成熟中間階段中（第5 - 6天；OPG- CTL) 或在蝕骨細胞成熟終段中（第7-15天；CTL-OPG)的蚀骨細胞形成檢定中加入。定量分析TRAP活性並與沒有OPG( CTL-CTL)而有OPG通過之情況中觀測到的活性比較（0PG-0PG)。圖22. IL-1卢，IL-1沈和OPG對於小鼠體内血液游離鈣的影響。在注射單獨的IL-1 /?，單獨的IL-1以，IL-1 yS+muOPG -13- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A 7 __B7 五、發明説明（11 )Figure 21. The effect of 0PG on the terminal differentiation of osteoclasts. Erosion of huOPG [22-401] _Fc fusion system in the middle stage of osteoclast maturation (days 5-6; OPG-CTL) or in the end stage of osteoclast maturation (days 7-15; CTL-OPG) Bone cell formation assay was added. The TRAP activity was quantified and compared with the activity observed in the absence of OPG (CTL-CTL) and OPG passage (0PG-0PG). Figure 22. Effects of IL-1 Lu, IL-1 Shen, and OPG on free calcium in blood in mice. Injecting separate IL-1 / ?, separate IL-1, IL-1 yS + muOPG -13- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) 1221482 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Printed by the consumer cooperative A 7 __B7 V. Description of the invention (11)

[22-401]_Fc，IL-1 從+mu〇PG[22-401]-Fc，及單獨的 mu〇PG[22-401] _Fc, IL-1 from + mu〇PG [22-401] -Fc, and mu〇PG alone

[22_401]-Fc之後監測血液游離鈣水平。對照小鼠係接受只有磷酸鹽緩衝食鹽水（PBS)的注射。A所示爲IL-1B實驗；B 所示爲IL-Ιπ實驗。圖2 3. OPG對於對照小鼠和IL1-處理小鼠體中頭頂蝕骨細胞之影響。分析小鼠頭頂骨樣品的組織學方法係在實施例11B 中説明。箭號所指爲在第2天處理過的小鼠體内所含的蝕骨細胞。（A)每天接受四次PBS注射，（B)每天一次IL-1注射和三次PBS注射，（C)每天注射一次PBS和三次OPG，（D)每天注射一次IL-1和三次〇PG的小鼠頭頂樣。圖24.在正常小鼠骨髓腔内的骨質蓄積之放射攝影分析。小鼠經皮下注射食鹽水（A)或muOPG[22-401]-Fc融合體（5毫克/公斤/天）1 4天（B)並依實施例11C中所述測定骨密度。圖2 5 ·正常小鼠骨髓腔中骨質蓄積之組織形態測定分析。注射實驗和骨組織學皆依實施例11C中所述實施。圖26·正常小鼠骨髓腔中骨質蓄積之組織學分析。注射實驗和骨組織學皆依實施例11C中所述實施。A·食鹽水注射。 B· muOPG[22_401]-Fc融合體注射。圖27.給到切除卵巢小鼠的蛋〇pg活性。於此兩週實驗中，減低骨密度的傾向顯然會被OPG或其他抗吸除性治療所阻斷。於卵巢切除時和在治療的第一週和第二週皆給予dexa 測量。其結果表爲相對於初始骨密度的❶/❶變化率（平均値+/_ SEM)。圖2 8 ·股幹骺端中的骨密度，以組織形態測定法測量者， -14 - 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X 297公釐） ----------------、τ------^ (請先閲讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 1221482 A7 B7 五、發明説明（12 ) 其在切除卵巢老鼠（OVX)中的値傾向於低於在卵巢切除後 1 7天的僞手術動物（SHAM)中的値。此效應會被OPG· Fc所阻斷，使得經〇PG-Fc處理過的卵巢切除老鼠（OVX+OPG)比經媒液處理的卵巢切除老鼠（OVX)具有明顯較高的骨密度（平均値+/-SEM)。發明之詳細説明本發明鑑別出一種新穎的腫瘤壞死因子受體（TNFR)超族的成員，其係從胎老鼠腸cDNA庫中離析出來的表現序列標籤（expressed sequence tag)(EST)。全長度老鼠 cDNA 純系和對應的小鼠與人類cDNA純系所具構造係依實施例1和6所述而測定的。老鼠，小鼠和人類諸基因分別表於圖2B-2C (SEQ ID NO : 120)，9A-9B((SEQ ID NO : 122)，和 9C-9D (SEQ ID NO : 124)之中。所有這三種序列都顯示出對TNFR 族員所具細胞外域的強烈相似性。該諸分離出的全長度 cDNA純系沒有一者有編碼對膜結合受體所預期的透膜域和細胞質域，可推測這些cDNAs係編碼可溶性，分泌蛋白質而非細胞表面受體。圖9D中所示跨越核甞酸1200-1353的一部份人類基因已在1995年11月22日登記在the Genebank的數據庫，登記號碼17188769。老鼠和人類mRNA的組織分佈係依實施例2中所述測定。在老鼠體内，於腎，肝，胎盤和心臟中偵檢mRNA表現而以腎中有最高的表現。此外也偵檢在骨骼肌和胰臟中的表現。於人體中，係在相同的組織以及淋巴節，胸腺，脾和闌尾内偵檢表現。 -15- 本紙張尺度_巾準（CNS ) A4規格（210X297公釐7 (請先閱讀背面之注意事項再填寫本頁)[22_401] -Fc monitor blood free calcium levels after. Control mice received injections of phosphate buffered saline (PBS) only. A shows the IL-1B experiment; B shows the IL-Iπ experiment. Figure 2 3. Effect of OPG on osteoclasts in the head of control and IL1-treated mice. Histological methods for analyzing mouse parietal bone samples are illustrated in Example 11B. The arrows indicate osteoclasts contained in mice treated on day 2. (A) received four PBS injections per day, (B) one IL-1 injection and three PBS injections per day, (C) one PBS injection and three OPG injections per day, and (D) one injection of IL-1 and three PG injections per day Mouse head-like. Figure 24. Radiographic analysis of bone accumulation in the bone marrow cavity of normal mice. Mice were injected subcutaneously with saline (A) or muOPG [22-401] -Fc fusion (5 mg / kg / day) for 14 days (B) and the bone density was determined as described in Example 11C. Figure 25. Histomorphological analysis of bone accumulation in the bone marrow cavity of normal mice. Both the injection experiment and bone histology were performed as described in Example 11C. Figure 26. Histological analysis of bone accumulation in the bone marrow cavity of normal mice. The injection experiments and bone histology were performed as described in Example 11C. A. Saline injection. B. muOPG [22_401] -Fc fusion injection. Figure 27. Opg activity given to ovariectomized mice. In this two-week experiment, the tendency to reduce bone mineral density was apparently blocked by OPG or other anti-absorption therapy. Dexa measurements were given at the time of ovariectomy and during the first and second weeks of treatment. The results are shown in the ❶ / ❶ change rate (mean 値 + / _ SEM) relative to the initial bone density. Figure 2 8 · Bone mineral density in the metaphysis of the femur, measured by histomorphometry, -14-This paper size applies the Chinese National Standard (CNS) A4 specification (21〇X 297 mm) ------ ----------, τ ------ ^ (Please read the notes on the back before filling out this page) Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 1221482 A7 B7 V. Description of the invention ( 12) Its pupae in ovariectomized mice (OVX) tend to be lower than pupae in pseudo-surgical animals (SHAM) 17 days after ovariectomy. This effect is blocked by OPG · Fc, so that oPG-Fc-treated ovariectomized mice (OVX + OPG) have significantly higher bone density than vehicle-treated ovariectomized mice (OVX) (mean 値+/- SEM). DETAILED DESCRIPTION OF THE INVENTION The present invention identifies a novel member of the tumor necrosis factor receptor (TNFR) superfamily, which is an expressed sequence tag (EST) isolated from a fetal mouse intestinal cDNA library. The full-length mouse cDNA clones and the corresponding mouse and human cDNA clones were constructed as described in Examples 1 and 6. Mouse, mouse and human genes are shown in Figures 2B-2C (SEQ ID NO: 120), 9A-9B ((SEQ ID NO: 122), and 9C-9D (SEQ ID NO: 124). All All three sequences showed strong similarities to the extracellular domains of members of the TNFR family. None of the isolated full-length cDNA clones had the transmembrane and cytoplasmic domains expected to encode membrane-bound receptors. These cDNAs encode soluble, secreted proteins rather than cell surface receptors. A portion of the human genes spanning nucleotides 1200-1353 shown in Figure 9D were registered in the Genebank database on November 22, 1995, with a registration number 17188769. Tissue distribution of mouse and human mRNA was determined as described in Example 2. In mice, mRNA performance was detected in kidney, liver, placenta, and heart, with the highest performance in kidney. In addition, detection was also performed. The performance in skeletal muscle and pancreas. In the human body, the same tissue and lymph node, thymus, spleen and appendix detection performance. -15- This paper size _ towel standard (CNS) A4 size (210X297 mm 7 (Please read the notes on the back before filling this page)

、1T —W· 1221482 經濟部中央標準局員工消費合作社印製 A7 —B7 五、發明説明（13 ) 老鼠cDNA係使用肝·特定性ApoE啓動基因表現系統在導入外來基因的小鼠體内表現（實施例3 )。表現子的分析顯示出骨密度有明顯地增加，特別是在長骨（股骨）。椎骨和扁平骨（骨盆骨）之間。骨經染色段的組織學分析顯示出嚴重的骨質石化病（參看實施例4)，顯示骨形成與吸除之間有顯著的不平衡，導致骨和軟骨的顯著蓄積。OPG表現子動物骨中小梁骨蝕骨細胞數目的減少顯示有明顯部份的TNFR-相關性蛋白質活性可能是在遏止骨吸除，一種由蚀骨細胞媒介的程序。從在導入外來基因的表現子中所具活性之觀點，將本文所述TNFR-相關性蛋白質稱爲〇PGs。使用老鼠cDNA序列，分離出小鼠和人類CDNA純系（實施例5 )。小鼠OPG在293細胞中及人類OPG在大腸桿菌中的表現分別在實施例7和8中説明之。小鼠〇PG係以Fc融合物形式產生，其係經蛋白質A親和性層析術純化的。此外也在實施例7中説全長度和截頭人類和小鼠〇pg多肽在CHO和293 細胞中的表現，成爲對人類IgGl的Fc區之融合多肽形式或爲未融合多肽。全長度和截頭人類和小鼠〇PGs在大腸桿菌内表現成爲F c融合多肽或爲未融合多肽，係在實施例8説明之。重組製成的哺乳動物和細菌〇PG的純化係在實施例1 〇中説明之。 OPG的生物活性係使用活體外（址^^)蚀骨細胞成熟檢定，間白素-l(IL-l)謗導高鈣血症活體内（汍模型，和正系小鼠骨岔度的注射研究予以測定的（參看實施例1丨）。下面在CHO或293細胞中產生的0PG重組蛋白質於大腸桿菌 -16- 冢紙張尺度適用中國國家標準（CNS ) A4規格（21〇><了97公釐） (請先閱讀背面之注意事項再填寫本頁)1T—W · 1221482 A7-B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (13) The mouse cDNA line uses the liver-specific ApoE promoter gene expression system to express in vivo mice introduced with foreign genes ( Example 3). Analysis of the expressors showed a significant increase in bone density, especially in long bones (femurs). Between the vertebra and the flat flat bone (pelvic bone). Histological analysis of the bone-stained section revealed severe osteopetrozoosis (see Example 4), showing a significant imbalance between bone formation and aspiration, resulting in significant accumulation of bone and cartilage. The reduction in the number of trabecular bone erosion bone cells in OPG-expressing animal bones shows that a significant portion of TNFR-related protein activity may be in the process of suppressing bone aspiration, a process mediated by bone erosion cells. From the viewpoint of their activity in the introduction of foreign genes, the TNFR-related proteins described herein are referred to as OPGs. Using mouse cDNA sequences, mouse and human CDNA clones were isolated (Example 5). The performance of mouse OPG in 293 cells and human OPG in E. coli is described in Examples 7 and 8, respectively. Mouse OPG was produced as an Fc fusion, which was purified by protein A affinity chromatography. Furthermore, in Example 7, the expression of full-length and truncated human and mouse Opg polypeptides in CHO and 293 cells was described as a fusion polypeptide form to the Fc region of human IgG1 or an unfused polypeptide. Full-length and truncated human and mouse OPGs appear as F c fusion polypeptides or unfused polypeptides in E. coli, as described in Example 8. Purification of recombinantly produced mammalian and bacterial OPG is described in Example 10. OPG's biological activity was measured using in vitro (site ^^) osteoclast maturation assays, and interleukin-1 (IL-1) was used to induce hypercalcemia in vivo (汍 model, and injection of orthopedic mice) It was determined by research (see Example 1 丨). The 0PG recombinant protein produced in CHO or 293 cells was applied to E. coli-16- mound paper scale in accordance with the Chinese National Standard (CNS) A4 specification (21〇>). 97 mm) (Please read the notes on the back before filling this page)

1221482 經濟部中央標準局員工消費合作社印製 A7 __B7 五、發明説明（14 ) 内蚀骨細胞成熟檢定中展現出活性：mu〇PG[22-185]-Fc， muOPG[22-194]-Fc，muOP<5[22-401]-Fc，muOPG[22-401]， huOPG[22-201]-Fc，huOPG[22-401]-Fc。在CHO細胞中產生的muOPG[22-180]-Fc和在大腸桿菌中產生的huOPG met[32· 401]於該活體外檢定中不展現出活性。有幾種來源的OPG係產生成爲二體物且於某些程度上成爲更高的多體物。在導入外來基因小鼠中產生的老鼠〇PG[22-401] ’在CHO細胞中產生成爲重組多肽的muOPGpa^Ol]和 huOPG[22-401]，以及從細胞毒性T細胞素表現成爲天然發生產物的OPG經在非還原性SDS凝膠上分析時大都爲二體物和二體物（參看實施例9)。在胺基酸186-401區中具有缺失的截頭OPG多肽（如〇pg[1-185]和OPG[l-194])大都爲單體物，可推測該186-401區可能參與OPG多肽的自我締合。不過，在大腸桿菌中產生的huOPG met [3 2-401]則大部份爲單體。 OPG在調節骨吸除作用中可能具有重要性。該蛋白質顯然係作爲TNF族的可溶性受體且可遏止包含在溶骨途徑中的受體-配體交互作用。該調節作用的一部份似乎爲蝕骨細胞數目的減少。核酸本發明提出編碼具有至少一種〇PG生物活性的多肽之離析核酸。如本文所述者，OPG生物活性包括，但不限於，包含骨新陳代謝的任何活性且特別者，包括增加骨密度。本發明的核酸係選自下列之中者： a)如圖 2B-2C(SEQ ID NO : 120)，9A-9B((SEQ ID NO : _ -17- 本紙張尺度賴巾咖家轉（c 遞格（2丨G χ 297公羡） (請先閱讀背面之注意事項再填寫本頁)1221482 Printed by A7 __B7 of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the Invention (14) The activity of maturation test of internal osteoblasts showed activity: mu〇PG [22-185] -Fc, muOPG [22-194] -Fc , MuOP < 5 [22-401] -Fc, muOPG [22-401], huOPG [22-201] -Fc, huOPG [22-401] -Fc. MuOPG [22-180] -Fc produced in CHO cells and huOPG met [32 · 401] produced in E. coli did not show activity in this in vitro assay. There are several sources of OPG lines that produce dimers and, to some extent, higher polymers. Mouse PG [22-401] 'produced in CHO cells to produce recombinant peptides muOPGpa ^ Ol] and huOPG [22-401] in CHO cells, and from cytotoxic T-cytokine to become naturally occurring The OPG of the product was mostly dimers and dimers when analyzed on a non-reducing SDS gel (see Example 9). The truncated OPG polypeptides (such as Opg [1-185] and OPG [1-194]) with deletions in the amino acid 186-401 region are mostly monomers. It can be speculated that the 186-401 region may participate in the OPG polypeptide Self-association. However, most of the huOPG met [3 2-401] produced in E. coli are monomers. OPG may be important in regulating bone resorption. This protein apparently acts as a soluble receptor in the TNF family and inhibits the receptor-ligand interactions involved in the osteolytic pathway. Part of this regulatory effect appears to be a reduction in the number of osteoclasts. Nucleic acid The present invention proposes an isolated nucleic acid encoding a polypeptide having at least one OPG biological activity. As described herein, OPG biological activity includes, but is not limited to, any activity that includes bone metabolism and, in particular, includes increasing bone density. The nucleic acid system of the present invention is selected from the following: a) as shown in Figures 2B-2C (SEQ ID NO: 120), 9A-9B ((SEQ ID NO: _-17- Passing (2 丨 G χ 297 public envy) (Please read the precautions on the back before filling this page)

、1T 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（15 ) 122)，和9C-9D((SEQ ID NO : 124)中所示的核酸序列或其互補股； b) 在嚴峻條件下與圖28-2(：((8£(51〇1^〇:120)，9八-9B((SEQ ID NO : 122)，和 9C_9D((SEQ ID NO : 124)中所示的多肽編碼區親合的核酸；及 c) 在嚴峻條件下與圖1A中所示核甞酸148至337(包括核甞酸148和337)段雜合的核酸；及 d )變性成（a)和(b)中所述序列的核酸序列。本發明提出編碼老鼠，小鼠和人類OPG的核酸以及雜合到彼等的核酸序列，彼等編碼具有至少一種OPG生物活性之多肽。此外也提出可雜合到涵蓋圖1 A中所示核甞酸148-337的老鼠OPG EST之核酸。雜合條件通常是高嚴緊性者例如 5xSSC，50%甲醯胺和42°C，爲本説明書實施例1中所述者。與這些條件同等嚴緊性者可以經由調整鹽和有機溶劑的濃度與溫度而易於得到。（b)中的核酸涵蓋著編碼著不會與其他已知的TNF受體超族成員發生可偵檢到的雜合之OPG-相關性多肽的核酸序列。於一較佳實施例中，該等核酸爲如圖 2B-2C((SEQ ID NO : 120)，9A-9B((SEQ ID NO : 122)，和 9C-9D((SEQ ID NO : 124)中所示者。本發明雜合核酸的長度係可變者，係因雜合可能發生於如圖 2B-2C((SEQ ID NO : 120)，9A-9B((SEQ ID NO : 122)，和 9C-9D((SEQ ID NO : 124)中所示編碼多肽的區域之部份或全部，且也可能發生於鄰接的非編碼區。所以，雜合核酸可爲圖 2B-2C((SEQ ID NO : 120)，9A-9B((SEQ ID NO : 122)， -18- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） (請先閲讀背面之注意事項再填寫本頁) 、?τ 1221482 A7 B7 五、發明説明（16 ) 和9C-9D((SEQ ID NO : 124)中所示序列的截頭者或延伸者。截頭或延伸核酸只要保留著一或多種OPG生物性質即涵蓋在本發明之内。該雜合核酸也可包括鄰接的非編碼區，彼等可爲OPG編碼區的5 ’端/或3 ’端。該等非編碼區包括參與 OPG表現的調節區，例如啓動基因，強化基因（enhance)，轉譯起始部位，轉錄終止部位和類似者。核酸的雜合條件載於 Sambrook et al. Molecular Cloning ： A Laboratory Manual- 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor，New York (1989)之中。經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 編碼老鼠OPG的DNA係摻加在質體pMO-Bl.l之中而於 1995 年 12 月 27 日保存在 the American Type Culture Collection, Rockville，MD其ATCC寄存號碼爲69970。編碼小鼠OPG的 DNA係摻加在質體pRcCMV•鼠類OPG之中而於1995年1 2月 27 日保存在 the American Type Culture Collection，Rockville， MD其寄存號碼爲69971。編碼人類OPG的DNA係摻加在質體 pRcCMV-人類OPG之中而於1995年12月27日保存在the American Type Culture Collection，Rockville，MD其登錄號碼爲69969。本發明核酸會在嚴緊條件下雜合到ATCC登錄號碼69969，69970和69971的DNA***體且具有至少一種OPG 生物活性。本發明也提出如圖2B，9 A和9B中所示的核酸序列之衍生物。如本文所用者，衍生物包括具有一或多個殘基的添加，取代，***或缺失之核酸序列使得所得序列所編碼的多肽具有一或多個經加入，缺失，插置或取代的胺基酸殘 -19- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（17 基且該所得多肽具有〇PG活性。該核酸衍生物可爲自然發生者，例如經由接合變異或多形現象，或可用熟諳此技者可取得的部位-導向突變形成技術予以構成者。自然發生的 OPG變體之一例子爲編碼前導序列中殘基3的丨丫丨變成之核酸（參看實施例5)。預期該等核酸衍生物係編碼分子中至少可此瓦解生物活性的區域内之胺基酸變化。其他衍生物包括編碼具有如圖 2B_2C((SEQ ID NO : 120)，9A-9B((SEQ ID NO : 122)，和 9C_9D((SEQ ID NO : 124)中所示細胞外域’及透膜域和細胞質域的膜結合形式〇PG之核酸。於一實施例中，OPG衍生物包括編碼著羧基端有一或多個胺基酸缺失掉的截頭形式OPG之核酸。編碼〇pg的核酸可從羧基端缺失掉1至216個胺基酸。視情況，可從新的羧基端延伸抗體F c區而得到具生物活性的〇pg_ fc融合多肽（參看實施例1 1)。於較佳實施例中，核酸係編碼著具有殘基22_ 185，22-189，22_194或22-201(使用圖9E-F中的編號）胺基酸序列之OPG且視情況也編碼一人類igG的F c區。此外也包括編碼著胺基端有一或多個胺基酸缺失掉的截頭形式OPG之核酸。截頭形式包括將構成前導序列的2 1個胺基酸之部份或全部都缺失掉者。另外，本發明提出編碼著從成熟胺基酸（在殘基22處）缺失掉1至10個胺基酸及，視情況，從羧基端（於殘基401處）缺失掉1至216個胺基的OPG 之核酸。視情況，該核酸可編碼在胺基端的甲硫胺酸殘基。彼等OPG截頭多肽的例子載於實施例8中。本發明核酸的例子包括cDNA，基因組DNA，合成DNA和 20- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（18 ) RNA。cDNA係得自從可表現OPG的各種組織分離出來的 mRNA製備成之庫。對於人類，OPG的組織來源包括胃，肝，胎盤和心。編碼OPG的基因組DNA係得自市售來自各種物種的基因組庫。合成DNA係經由將重疊寡核甞酸片段化學合成後組合各片段以重構成部份或全部的編碼區和側翼序列而得者（參看美國專利第4,695,623號所述干擾素基因的化學合成）。RNA可經由導引高水平mRNA合成的原核生物表現媒體，例如使用T 7啓動基因和RNA聚合酶的媒體而最容易地得到。本發明核酸序列可用來偵檢生物樣品中的OPG序列以測定何種細胞和組織可表現OPG mRNA。該等序列也可以用來針對OPG相對性序列篩選cDNA庫和基因組庫。彼等筛選係諳於此技者能力所及者，可以利用恰當的雜合條件來偵檢同系序列。該等核酸也可以用來經由反意治療或基因治療而調制OPG表現水平。該等核酸也可用來發育導入外來基因的動物以用來生產該多肽及用於生物活性的研究（參看實施例 3” 媒體和宿主細胞本發明也提出含有編碼OPG的核酸之表現媒體，用該媒體轉形過的宿主細胞及生產OPG的方法。重組蛋白質表現的評述載於 Methods of Enzymologv v 185，Goeddel，D. V. ed. Academic Press (1990) o 生產OPG的宿主細胞包括原核生物宿主細胞，例如大腸样菌，酵母菌，植物，昆蟲和哺乳動物宿主細胞。大腸桿菌 -21 - 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） (請先閱讀背面之注意事項再填寫本頁)1T 1221482 A7 B7 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the Invention (15) 122), and the nucleic acid sequence shown in 9C-9D ((SEQ ID NO: 124) or its complement); b) Under severe conditions with Figure 28-2 (: ((8 £ (51〇1 ^ 〇: 120), 9-8-9B ((SEQ ID NO: 122), and 9C_9D ((SEQ ID NO: 124) A nucleic acid that is affinity for the polypeptide coding region shown; and c) a nucleic acid that hybridizes with nucleotides 148 to 337 (including nucleotides 148 and 337) shown in FIG. 1A under severe conditions; and d) denatures ( The nucleic acid sequences of the sequences described in a) and (b). The present invention proposes nucleic acids encoding mouse, mouse and human OPG and hybridized nucleic acid sequences to them, which encode a polypeptide having at least one biological activity of OPG. Nucleic acids that can be hybridized to mouse OPG EST covering nucleotides 148-337 shown in Figure 1 A are also proposed. The hybridization conditions are usually high stringency such as 5xSSC, 50% formamidine, and 42 ° C. It is described in Example 1 of the specification. Those with the same stringency as these conditions can be easily obtained by adjusting the concentration and temperature of the salt and the organic solvent. The nucleic acid in (b) covers The nucleic acid sequence of the detectable hybrid OPG-related polypeptide will not occur with other known members of the TNF receptor superfamily. In a preferred embodiment, the nucleic acids are shown in Figures 2B-2C. ((SEQ ID NO: 120), 9A-9B ((SEQ ID NO: 122), and 9C-9D ((SEQ ID NO: 124)). The length of the hybrid nucleic acid of the present invention is variable, The heterozygosity may occur as shown in Figures 2B-2C ((SEQ ID NO: 120), 9A-9B ((SEQ ID NO: 122), and 9C-9D ((SEQ ID NO: 124)) Some or all of the regions may occur in adjacent non-coding regions. Therefore, the hybrid nucleic acid can be as shown in Figure 2B-2C ((SEQ ID NO: 120), 9A-9B ((SEQ ID NO: 122) -18- This paper size applies Chinese National Standard (CNS) A4 specification (210 × 297 mm) (Please read the precautions on the back before filling out this page),? Τ 1221482 A7 B7 V. Description of Invention (16) and 9C- A truncated or extended sequence of the sequence shown in 9D ((SEQ ID NO: 124). A truncated or extended nucleic acid is encompassed within the invention as long as it retains one or more of the OPG biological properties. The hybrid nucleic acid may also include Contiguous non-coding region They may be the 5 'end or the 3' end of the OPG coding region. Such non-coding regions include regulatory regions involved in OPG expression, such as promoter genes, enhancement genes, translation initiation sites, transcription termination sites, and the like. Nucleic acid hybridization conditions are described in Sambrook et al. Molecular Cloning: A Laboratory Manual- 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). The DNA encoding the mouse OPG was incorporated into plastid pMO-Bl.l and saved on December 27, 1995. The American Type Culture Collection, Rockville, MD has an ATCC deposit number of 69970. The DNA encoding the mouse OPG was incorporated into the plastid pRcCMV • mouse OPG and was deposited in the American Type Culture Collection, Rockville, MD on February 27, 1995. Its deposit number is 69971. The DNA encoding human OPG was incorporated into the plastid pRcCMV-human OPG and was stored in the American Type Culture Collection, Rockville, MD on December 27, 1995. Its registration number was 69969. The nucleic acid of the present invention is hybridized to the DNA inserts of ATCC accession numbers 69969, 69970, and 69971 under stringent conditions and has at least one OPG biological activity. The invention also proposes derivatives of the nucleic acid sequences as shown in Figures 2B, 9 A and 9B. As used herein, derivatives include nucleic acid sequences with the addition, substitution, insertion or deletion of one or more residues such that the polypeptide encoded by the resulting sequence has one or more amino groups added, deleted, inserted or substituted. Acid Residue-19- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (17 bases and the resulting peptide has 〇PG activity The nucleic acid derivative can be naturally occurring, for example, via splice mutation or polymorphism, or it can be constructed using site-directed mutation formation techniques available to those skilled in the art. An example of a naturally occurring OPG variant is coding The nucleic acid of residue 3 in the leader sequence (see Example 5). It is expected that these nucleic acid derivatives are amino acid changes in the region encoding the molecule that can at least disrupt the biological activity. Other derivatives include coding It has extracellular domains' and transmembrane domains and cytoplasm as shown in Figures 2B_2C ((SEQ ID NO: 120), 9A-9B ((SEQ ID NO: 122), and 9C_9D ((SEQ ID NO: 124) Nucleic acid of the membrane-bound form OPG. In one embodiment, the OPG derivative includes a nucleic acid encoding a truncated form of OPG with one or more amino acids deleted at the carboxyl terminus. The nucleic acid encoding Opg may be deleted from the carboxyl terminus. 1 to 216 amino acids can be dropped. Optionally, the antibody F c region can be extended from the new carboxy terminus to obtain a biologically active 0pg_fc fusion polypeptide (see Example 1 1). In a preferred embodiment, the nucleic acid system It encodes the OPG with the amino acid sequence of residues 22_185, 22-189, 22_194 or 22-201 (using the numbers in Figures 9E-F) and optionally also encodes the F c region of a human igG. It also includes a coding Nucleic acid of one or more truncated forms of OPG at the amino terminus. The truncated form includes those in which some or all of the 21 amino acids constituting the leader are deleted. In addition, the present invention It is proposed to encode an OPG that deletes 1 to 10 amino acids from the mature amino acid (at residue 22) and, optionally, 1 to 216 amino groups from the carboxy terminus (at residue 401). Nucleic acid. Optionally, the nucleic acid can encode a methionine residue at the amine end. Their OPG Examples of noggin are given in Example 8. Examples of the nucleic acids of the present invention include cDNA, genomic DNA, synthetic DNA and 20- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the back first Note: Please fill in this page again.) Order 1221482 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of invention (18) RNA. CDNA is a library prepared from mRNA isolated from various tissues that can express OPG. For humans, tissue sources of OPG include stomach, liver, placenta, and heart. The genomic DNA encoding OPG was obtained from commercially available genomic libraries from various species. Synthetic DNA is obtained by chemically synthesizing overlapping oligonucleotide fragments and combining the fragments to reconstruct part or all of the coding region and flanking sequences (see the chemical synthesis of the interferon gene described in US Patent No. 4,695,623). RNA is most easily obtained via prokaryotic expression media that direct high-level mRNA synthesis, such as media using the T7 promoter and RNA polymerase. The nucleic acid sequences of the invention can be used to detect OPG sequences in biological samples to determine which cells and tissues can express OPG mRNA. These sequences can also be used to screen cDNA and genomic libraries for OPG relative sequences. Their screening is within the skill of the artist, and they can detect homologous sequences using appropriate heterozygous conditions. These nucleic acids can also be used to modulate OPG expression levels via anti-sense therapy or gene therapy. These nucleic acids can also be used to develop animals into which foreign genes have been introduced for the production of the polypeptide and for the study of biological activity (see Example 3). Media and Host Cells The present invention also provides expression media containing a nucleic acid encoding OPG. Media-transformed host cells and methods for producing OPG. A review of the performance of recombinant proteins is contained in Methods of Enzymologv v 185, Goeddel, DV ed. Academic Press (1990) o OPG-producing host cells include prokaryotic host cells, such as the large intestine Like bacteria, yeasts, plants, insects and mammalian host cells. E. coli-21-This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the precautions on the back before filling this page )

、1T -麯· 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（19 ) 菌株例如HB101或JM101即適合於表現之用。較佳的哺乳動物宿主細胞包括COS，CHOd-，293，CV_1，3T3，倉鼠嬰腎 (BHK)細胞和其他。在宿主一轉譯修飾，例如糖基化作用和多肽處理修飾，對於OPG活性具重要性之時，以哺乳動物宿主細胞較適當。哺乳動物表現可促成可以從生長培養基回收得到分泌多肽之產生。用於OPG表現的媒體含有媒體繁殖和選殖***體表現所需的最少序列。這些序列包括複製源，選擇標記，啓動基因，核糖體結合部位，強化子序列，RNA接合部位和轉錄終止部位。適合於在前述宿主細胞内表現的媒體皆爲易於取得者且本發明的核酸係用標準重組DNA技術插到媒體内的。此外也包括組織特定性OPG表現用之媒體。彼等媒體包括可在肝，腎或其他器官中特定地作用供在小鼠内生產用之啓動基因，及供標的人類細胞内進行OPG表現所用之病毒媒體。使用恰當的宿主·媒體系統，經由將經含有編碼OPG的核酸之表現媒體轉形過宿主細胞在產生OPG的條件下培養，及分離出表現產物即可重組地產生OPG。OPG係產生在經轉染哺乳動物細胞的上澄液内或在轉形細菌宿主細胞的包涵體内。如此產生的OPG可由諳於此技者依下文所述程序予以純化。在哺乳動物與細菌宿主系統内的OPG表現係在實施例7 和8中説明之。哺乳動物宿主的表現媒體可用質體例如在 PCT申請第90/14363中所述的pDSR從爲例説明之。細菌宿主細胞的表現媒體之例子有實施例8中所述的質體pAMG21和 -22- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁)1T-Qu 1221482 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (19) Strains such as HB101 or JM101 are suitable for performance purposes. Preferred mammalian host cells include COS, CHOd-, 293, CV_1, 3T3, hamster infant kidney (BHK) cells and others. When host-translational modifications, such as glycosylation and polypeptide processing modifications, are important for OPG activity, mammalian host cells are more appropriate. Mammalian manifestations can contribute to the production of secreted polypeptides that can be recovered from growth media. The media used for OPG expression contains the minimum sequence required for media reproduction and selection of insert insertions. These sequences include sources of replication, selectable markers, promoter genes, ribosome binding sites, enhancer sequences, RNA junction sites, and transcription termination sites. All media suitable for expression in the aforementioned host cells are readily available and the nucleic acid system of the present invention is inserted into the media using standard recombinant DNA technology. It also includes media for organizing specific OPG performance. These media include viral media that can specifically act in the liver, kidney, or other organs for starter genes for production in mice, and viral media for OPG expression in target human cells. Using an appropriate host-media system, OPG can be produced recombinantly by transforming host cells transformed with expression media containing a nucleic acid encoding OPG under conditions that produce OPG and isolating the expression products. OPG lines are produced in supernatants of transfected mammalian cells or in inclusion bodies of transformed bacterial host cells. The OPG thus produced can be purified by those skilled in the art in accordance with the procedure described below. The expression of OPG in mammalian and bacterial host systems is illustrated in Examples 7 and 8. Mammalian host expression media can be exemplified by plastids such as those described in PCT application 90/14363. Examples of expression media for bacterial host cells are pAMG21 and -22 described in Example 8. This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the precautions on the back before (Fill in this page)

、1T -婣· 1221482 A7 B7 五、發明説明（2〇 ) pAMG22-His。質體pAMG21已在1996年7月24曰寄存在the American Type Culture Collection，Rockville，MD，其登錄號碼爲98113。質體pAMG22-His則在1996年7月24曰寄存在the American Type Culture Collection，Rockville，MD，其登錄號碼爲98112。要預先説明者，所述及的特殊質體和宿主細胞皆爲供闡述目的者，其他可取得的質體和宿主細胞也可以用來表現出該多肽。本發明也提出以内源性核酸經由活體内或活體外重組事件表現出OPG以促成來自宿主染色體的OPG調制。此外本發明也涵蓋經由導入能夠導引從内源性OPG密碼區生產OPG的外源性調節序列（如啓動基因或強化因子）而進行之OPG表現。另外本發明也提出對能夠導引OPG生產的内源性調節序列之刺激方法（例如經由暴露於轉錄強化因子）。多肽經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 本發明提出OPG，一種新穎的TNF受體超族成員，其具有與骨新陳代謝相關聯的活性且特別者具有抑制骨質吸除藉以增加骨密度之活性。OPG指的是具有至少一種OPG生物活性的小鼠，老鼠或人類OPG或其衍生物的胺基酸序列之多肽。老鼠，小鼠和人類OPG的胺基酸序列分別示於圖IB-SCGSEQ ID NO : 121) ， 9A-9B((SEQ ID NO : 123) ，和9(：· 9D((SEQ ID NO : 125)之中。OPG衍生物指的是有添加，缺失，***或取代一或多個胺基酸使得所產生的多肽具有至少一種OPG生物活性之多肽。OPG生物活性包括，但不限於，涉及骨新陳代謝之活性。較佳者，該多肽係將2 1個胺 -23- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 __B7 五、發明説明（21 ) 基酸的胺基末端前導序列脱除掉者。本發明所涵蓋的OPG多肽包括老鼠[1-40]，老鼠[22-180]，老鼠[22-401]，老鼠[22-401]-Fc融合物，老鼠[l_180]-Fc融合物，小鼠[1-401]，小鼠[1-180]，小鼠[22-401]，人類[1-401]，小鼠[22-180]，人類[22_401]，人類[22-180]，人類[1-180]，人類[22-180]-Fc融合物和人類met-32-401。胺基酸編號係 SEQ ID NO : 121(老鼠），SEQ ID NO : 123(小鼠)和SEQ ID NO : 125(人類）中所示者。另外也涵蓋多肽衍生物。其將OPG的胺基酸殘基180-401部份或全部地缺失或羧基端截頭；於殘基180·401中有一或多個胺基酸改變；OPG的富含半胱胺酸或部份或全部地缺失掉，特別是遠端（羧基端）富含半胱胺酸域之缺失；及富含半胱胺酸域，特別是遠端（羧基端）富含半胱胺酸域中有一或多個胺基酸改變。於一實施例中，OPG係將其羧基端的1至約216胺基酸缺失掉。於另一實施例中，OPG係從其成熟胺基端缺失掉1至約10胺基酸 (其中該成熟胺基端係位於殘基2 2 )且視情況，從其羧基端缺失掉1至約216胺基酸。本發明所涵蓋的其他OPG多肽包括下列：人類[22-180]-Fc 融合體，人類[22_201]-Fc融合體，人類[22-401]-Fc融合體，小鼠[22-185]-Fc融合體，小鼠[22_194]-Fc融合體。這些多肽係在哺乳動物宿主的細胞，例如CHO或293細胞中產生的。本發明所涵蓋的在原核生物宿主細胞内表現之其他OPG多肽包括下列：人類met[22-401]，Fc-人類met[22-401]融合體（fc 區係融合在全強度OPG密碼序列的胺基端，如實施例8中所 -24- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂丨續· 1221482 A7 B7 五、發明説明（22 ) 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 述者），人類met[22-401]-Fc融合體（Fc區係融合到全長度 OPG序列），Fc_ 小鼠 met[22-401]融合體，小鼠met[22-401]-融合體，人類met[27_401]，人類 met[22-185]，人類met[22-189]，人類 met[22-194]，人類 met[22_194](P25A)，人類 met[22-194](P26A)，人類 met[27-185]，人類 met[27-189]，人類 met[27-194]，人類 met-arg-gly-ser-(his)6[22-401]，人類 met-lys[22-401]，人類met-(lys)3-[22-401]，人類 met[22-401]-Fc(P25A)，人類 met[22-401](P25A)，人類 met[22-401] (P26A)，人類 met[22-401](P26D)，小鼠met[22-401]，小鼠 met[27_401]，小鼠 met[32-401]，小鼠 met[22-180]，小鼠 met[22_189]，小鼠 met[22_194]，小鼠 met[27-189]，小鼠 met[27-194]，小鼠 met-lys[22-401]，小鼠 ΗΕΚ[22·401] (Α45Τ)，小鼠 met-lys-(his)7[22-401]，小鼠 met-lys[22-401p (his)7和小鼠 met[22-401](P33E，G36S，A45P)。要了解上述在原核生物宿主細胞内產生的OPG多肽都具有一個胺基端甲硫胺酸殘基，即使此種殘基未示出亦然。於特殊實施例子中係使用具有 Ellison et al.(Nuc. Acids Res. !〇_，4071-4079(1982))中所示序列的人類IgGl· r 1所含227胺基酸區來產生OPG-Fc融合體。不過，也可以使用人類IgG所含Fc區的變體。融合到人類IgGl Fc區的羧基端OPG截頭物所具生物活性之分析顯示出一活性所需的約164胺基酸之OPG部份。此區涵蓋著胺基酸22-185，較佳者爲9C-9D((SEQ ID NO : 125)中者，且包括四個具有TNFR細胞外域所含富含半胱胺酸域特 -25- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（23 ) 性的富含半胱胺酸域。使用OPG與TNF受體族員所含細胞外配體結合域之間的同系性，根據TNFR-1所含細胞外域的已知結晶構造產生〇pg 的三次元模型（參看實施例6)。使用此模型來鑑別〇PG内對生物活性具重要性之殘基。鑑別出參與維持該四個富含半胱胺酸域的構造之半胱胺酸殘基。在模型中鑑別出下列雙硫鍵：域1 : cys41 對 cys54，cys44對cys62，tyr23和his66可用來安定此域的構造；域2 ·· cys65對cys80，cys83對cys98， cys87對cysl05 ;域3 ·· cysl07對 cysll8，cysl24對cysl42 ;域 4 : cysl45對cyl060，cysl66對cysl85。此外也鑑別出密切靠近TNF Θ的殘基如圖1 1和12A_12B中所示者。於此模型中，假設OPG係結合到對應的配體；使用TNF/?爲模型配體以刺激OPG與其配體之交互作用。根據此模型化，〇pg中的下列殘基可能對配體結合具有重要性：glu34，lys43，pro66至 gln91(特別者，pro66，his68，tyr69，tyr70，thr71，asp72， ser73 ’ his76 ， ser77 ， asp78 ， glu79 ， leu81 ， tyr82 ， pro85 ， val86 ’ lys88，glu90和 gln91)，glul53及serl55 o 這些胺基酸殘基的變更，單獨地或組合地，都可能變更 OPG的生物活性。例如，特定光胱胺酸殘基的改變可能變更個別富含半胱胺酸域的構造，而對配體結合具重要性的殘基之變化可能影響〇PG與配體的物理交互作用。構造模型可能幫助鑑定出具有更良好性質的類似物，例如具有增強的生物活性，更大的安定性，或更大的調配容易性者。本發明也提出，由〇PG單體構成的〇PG多體物。OPG似乎 __— -26- 本紙張尺度適用中國國家標準（CNS ) A4規格（2lGx297公瘦） 1· 11 n I ϋ I n ϋ I ^ (請先閲讀背面之注意事項再填寫本頁) 1221482 A7 B7 五、發明説明（24 ) 是以多體物來作用者（例如，二體物，三體物或更高數目的單體物者）。較佳者，該〇PG多體物爲二體物或三體物。 OPG多體物可包括具有足以促進多體物形成的01>(3胺基酸序列之單體或可包括具有異系序列例如抗體Fc區之單體。〇PG 羧基端缺失之分析可推得在區186-4〇1中至少有一部份參與 OPG多肽的缔合。將〇pG胺基酸186-401區的部份或全部以能夠自缔合的胺基酸序列予以取代也是涵蓋在本發明之内者。另外，OPG多肽或其衍生物可經修飾以形成二體物或多體物，如經由部位導引突變形成以造成未配對半胱胺酸殘基供鏈間雙硫鍵形成所用，經由光化學交聯，例如曝光到紫外光，或經由用雙官能性聯結子分子如雙官能聚乙二醇和類似者進行化學交聯。本發明也涵蓋OPG多肽的修飾且其包括後-轉譯修飾（如， N -鍵聯或〇 -鍵聯的醣鏈，N-端或C-端的修飾處理），將化學部份體連接到胺基酸主鏈，N -鍵聯或〇 -鍵聯醣鏈的化學修飾，及因原核生物宿主細胞表現的結果所致N_端甲硫胺酸殘基之添加。該多肽也可以用可偵檢標記予以修舞，例如用酵素性，螢光性，同位素或親和性標記修飾以供蛋白質偵檢和分離之用。經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) -l-tv 其他的OPG修飾包括OPG融合到異系胺基酸序列所形成的歆合型蛋白質。该異系序列可爲能促使所得融合蛋白質保留住0PG活性之任何序列。該等異系序列包括例如，免疫球蛋白融合體’例如Fc融合體’有助於蛋白質的純化。較佳者爲能促進OPG單體缔合形成二體物，三體物和其他更高的 -27 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（25 ) ~ ~ 多體物形式之異系序列。本發明多肽係從表現出OPG的組織，細腧玄i从 & +飑系和轉形宿主細胞中所含的其他多肽中分離純化出，或從仝 .,^ 含有分泌蛋白質的細胞培養物中所含成份純化出。於一實施例中，該多肤未與其他人類蛋白質，例如細菌宿主細胞的表現產物。本發明也提出經化學修飾的OPG衍生物，彼等可提^附加的優點例如增加多肽的安定性和循環時間，或減低免疫^ 傳性（參看美國專利第4，179,337號）。衍化所用化學部份體可選自水溶性聚合物例如聚乙二醇，乙二醇/丙二醇共^ 物，羧甲基纖維素，葡聚糖，聚乙烯醇和類似者:該/多肽可在分子内的隨機位置，或在分子内的預定位置修飾且可包括一’二，三或更多個接著的化學部份體。該聚合物可爲具有任何分子量者，且可爲分支或未分枝者。對於聚乙二醇而言，較佳的分子量爲介於約lkDa=約 lOOkDa之間（"約"一詞表示在聚乙二醇的製備中，某些分子可能比所述分子量較重或較輕）以使處理和製造較爲容易。依所欲治療剖型而定，可以使用其他的大小（如根據所欲持續放的持續期，若有時，對生物活性的影響，處理的容易性，抗原性的程度或缺乏及聚乙二醇對治療性蛋白質或類似物的其他已知影響）。聚乙二醇分子（或其他化學部份體）必須在考慮到對蛋白質官能域或抗原域的影響之下接著到蛋白質上。有許多接著方法可供諳於此技者所用，如EP 〇 401 384中所述者，其併於本文作爲參考（將PEG偶合到G-CSF)，也參看Malik et al. -28- 氏張尺度適用中國國家標準（CNS) 丨Gx297公着） (請先閲讀背面之注意事項再填寫本頁) 、?τ 經濟部中央標準局員工消費合作社印製 1221482 A7 B7 五、發明説明（26 )1T- 婣 1221482 A7 B7 V. Description of the invention (20) pAMG22-His. The plastid pAMG21 was deposited at the American Type Culture Collection, Rockville, MD on July 24, 1996, and its registration number is 98113. The plastid pAMG22-His was deposited at the American Type Culture Collection, Rockville, MD on July 24, 1996, and its registration number was 98112. To be stated in advance, the specific plastids and host cells mentioned are for the purpose of illustration, and other available plastids and host cells can also be used to express the polypeptide. The invention also proposes that OPG is expressed by endogenous nucleic acid via in vivo or ex vivo recombination events to facilitate OPG modulation from the host chromosome. In addition, the present invention also covers OPG performance by introducing exogenous regulatory sequences (such as a promoter or an enhancement factor) that can guide the production of OPG from an endogenous OPG code region. In addition, the present invention also proposes a method of stimulating endogenous regulatory sequences capable of directing OPG production (e.g., through exposure to a transcription-enhancing factor). Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Peptide Economics (please read the precautions on the back before filling this page) The present invention proposes OPG, a novel member of the TNF receptor superfamily, which has activity related to bone metabolism and is particularly It has the activity of inhibiting bone resorption to increase bone density. OPG refers to a peptide of the amino acid sequence of mouse, mouse or human OPG or a derivative thereof having at least one biological activity of OPG. The amino acid sequences of mouse, mouse and human OPG are shown in Figures IB-SCGSEQ ID NO: 121), 9A-9B ((SEQ ID NO: 123), and 9 (: · 9D ((SEQ ID NO: 125 ). OPG derivative refers to a polypeptide that has the addition, deletion, insertion or substitution of one or more amino acids so that the resulting polypeptide has at least one OPG biological activity. OPG biological activity includes, but is not limited to, bone Metabolic activity. Preferably, the peptide will be 21 amines. 23- This paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 mm) 1221482 Printed by A7 __B7 5. Description of the invention (21) Those whose amino acid terminal leader sequence is removed. The OPG polypeptides covered by the present invention include mouse [1-40], mouse [22-180], mouse [22-401], mouse [22-401] -Fc fusion, mouse [l_180] -Fc fusion, mouse [1-401], mouse [1-180], mouse [22-401], human [1-401], Mouse [22-180], human [22_401], human [22-180], human [1-180], human [22-180] -Fc fusion and human met-32-401. Amino acid numbering system SEQ ID NO: 121 ( (Mouse), SEQ ID NO: 123 (mouse) and SEQ ID NO: 125 (human). Polypeptide derivatives are also encompassed. They partially or fully include amino acid residues 180-401 of OPG Deletion or truncation at the carboxy terminus; one or more amino acid changes in residue 180 · 401; cysteine-rich or partial or complete deletion of OPG, especially the distal (carboxy-terminus) -rich Deletion of a cysteine domain; and one or more amino acid changes in a cysteine-rich domain, especially a distal (carboxy-terminal) cysteine-rich domain. In one embodiment, OPG is 1 to about 216 amino acids at the carboxyl terminus are deleted. In another embodiment, OPG is 1 to about 10 amino acids (from the mature amino terminus at residue 2) deleted from its mature amino terminus. 2) and optionally, 1 to about 216 amino acids are deleted from its carboxy terminus. Other OPG polypeptides covered by the present invention include the following: human [22-180] -Fc fusions, human [22_201] -Fc fusions , Human [22-401] -Fc fusion, mouse [22-185] -Fc fusion, mouse [22_194] -Fc fusion. These polypeptides are in mammalian host cells, such as C Produced in HO or 293 cells. Other OPG polypeptides expressed in prokaryotic host cells covered by the present invention include the following: human met [22-401], Fc-human met [22-401] fusion (fc region Fused to the amine base of the full-strength OPG code sequence, as described in Example -24. This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the precautions on the back before filling in this Page) Order 丨 continued · 1221482 A7 B7 V. Description of Invention (22) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the precautions on the back before filling out this page) (narrator), human met [22-401] -Fc fusion (the Fc region is fused to a full-length OPG sequence), Fc_ mouse met [22-401] fusion, mouse met [22-401] -fusion, human met [27_401], human met [22 -185], human met [22-189], human met [22-194], human met [22_194] (P25A), human met [22-194] (P26A), human met [27-185], human met [27-189], human met [27-194], human met-arg-gly-ser- (his) 6 [22-401], human met-lys [22-401], human met- (lys) 3 -[22-401], human met [22-401] -Fc (P25A), human met [22-401] (P25A), human met [22-401] (P26A), human met [22-401] (P26D), mouse met [22-401], mouse met [27_401], mouse met [32-401], mouse met [22-180], mouse met [22_189], mouse met [22_194], mouse met [27-189], mouse met [ 27-194], mouse met-lys [22-401], mouse ΗΕΚ [22 · 401] (Α45Τ), mouse met-lys- (his) 7 [22-401], mouse met-lys [ 22-401p (his) 7 and mouse met [22-401] (P33E, G36S, A45P). It is to be understood that the above-mentioned OPG polypeptides produced in a prokaryotic host cell all have an amino terminal methionine residue, even if such a residue is not shown. In a specific implementation example, the 227 amino acid region contained in human IgGl · r 1 having the sequence shown in Ellison et al. (Nuc. Acids Res.! 〇_, 4071-4079 (1982)) was used to generate OPG- Fc fusion. However, variants of the Fc region contained in human IgG can also be used. Analysis of the biological activity of the carboxy-terminated OPG frustum fused to the Fc region of human IgGl revealed an OPG moiety of approximately 164 amino acids required for activity. This region covers amino acids 22-185, preferably 9C-9D ((SEQ ID NO: 125)), and includes four cysteine-rich domains containing TNFR extracellular domain-specific 25- This paper size applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (23) A cysteine-rich domain. Using OPG The homology with the extracellular ligand-binding domain contained in the TNF receptor family members is based on the known crystal structure of the extracellular domain contained in TNFR-1. A three-dimensional model of 0 pg is generated (see Example 6). This model is used. To identify residues important to biological activity in OPG. Identified cysteine residues that are involved in maintaining these four cysteine-rich domain constructs. The following disulfide bonds were identified in the model: domain 1: cys41 to cys54, cys44 to cys62, tyr23 and his66 can be used to stabilize the structure of this domain; domain 2 · cys65 to cys80, cys83 to cys98, cys87 to cysl05; domain 3 · cysl07 to cysll8, cysl24 to cysl42; domain 4: cysl45 to cyl060, cysl66 to cysl85. In addition, it is identified The residues of TNF Θ are shown in Figures 11 and 12A-12B. In this model, it is assumed that OPG is bound to the corresponding ligand; TNF /? Is used as a model ligand to stimulate the interaction between OPG and its ligand. According to In this modelling, the following residues in Opg may be important for ligand binding: glu34, lys43, pro66 to gln91 (in particular, pro66, his68, tyr69, tyr70, thr71, asp72, ser73 'his76, ser77, asp78 , Glu79, leu81, ty82, pro85, val86 'lys88, glu90 and gln91), glul53 and serl55 o Changes in these amino acid residues, alone or in combination, may change the biological activity of OPG. For example, specific photocysteine Changes in amino acid residues may change the structure of individual cysteine-rich domains, and changes in residues important for ligand binding may affect the physical interaction of PG with the ligand. Structural models may help identify Analogs with better properties, such as those with enhanced biological activity, greater stability, or greater ease of deployment. The present invention also proposes an PG polymer consisting of PG monomers. OPG It seems __— -26- This paper size applies to Chinese National Standard (CNS) A4 (2lGx297 male thin) 1 · 11 n I ϋ I n ϋ I ^ (Please read the precautions on the back before filling this page) 1221482 A7 B7 V. Description of the invention (24) Those who act on multiple bodies (for example, those with two bodies, three bodies or higher number of monomers). Preferably, the OPG polysome is a dibody or a tribody. OPG multimers may include monomers with a sufficient amino acid sequence of <3> (3 amino acids) or may include monomers with heterologous sequences such as the Fc region of an antibody. Analysis of the carboxy-terminal deletion of PG can be deduced At least a part of region 186-4〇1 is involved in the association of OPG polypeptides. It is also encompassed in the present invention that a part or all of the amino acid region 186-401 of opG is replaced with an amino acid sequence capable of self-association. Within the invention. In addition, OPG polypeptides or derivatives thereof can be modified to form dimers or multimers, such as by site-directed mutation formation to cause formation of disulfide bonds between the unpaired cysteine residue donor chains. Used, via photochemical cross-linking, such as exposure to ultraviolet light, or via chemical cross-linking with bifunctional linker molecules such as bifunctional polyethylene glycol and the like. The invention also encompasses modifications of OPG polypeptides and includes post- Translational modifications (eg, N-linked or O-linked sugar chains, N- or C-terminal modification processing), linking chemical moieties to amino acid backbones, N-linked or O-bonds Chemical modification of linked sugar chains, and the result of the performance of prokaryotic host cells Addition of N-terminal methionine residues. The peptide can also be modified with detectable labels, such as modified with enzyme, fluorescent, isotope or affinity labels for protein detection and isolation Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) -l-tv Other OPG modifications include conjugated proteins formed by fusion of OPG to heterologous amino acid sequences. The heterologous sequence can be any sequence that can cause the resulting fusion protein to retain OPG activity. Such heterologous sequences include, for example, immunoglobulin fusions such as Fc fusions, which facilitate protein purification. The preferred is Promote the association of OPG monomers to form dimers, trisomys and other higher -27 1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, printed A7 B7 V. Description of the invention (25) ~ ~ Diversity of multibody forms Sequence: The polypeptide of the present invention is isolated from tissues exhibiting OPG, and is purified from other polypeptides contained in & + lineage and transformed host cells, or from cells containing secreted proteins. The ingredients contained in the nutrient are purified. In one embodiment, the polypeptide is not a product of expression with other human proteins, such as bacterial host cells. The present invention also proposes chemically modified OPG derivatives, which can be extracted and added. Advantages such as increasing the stability and circulation time of the polypeptide, or reducing the immunogenicity (see US Pat. No. 4,179,337). The chemical moiety used for the derivatization can be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol Alcohol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like: The / polypeptide can be modified at random positions within the molecule, or at predetermined positions within the molecule and can include one, two, three Or more subsequent chemical moieties. The polymer may be of any molecular weight and may be branched or unbranched. For polyethylene glycol, a preferred molecular weight is between about lkDa = about 100 kDa (" about " means that in the preparation of polyethylene glycol, certain molecules may be heavier than the molecular weight Or lighter) to make handling and manufacturing easier. Depending on the profile to be treated, other sizes can be used (eg according to the duration of the desired continuous release, if sometimes, the effect on biological activity, ease of handling, degree or lack of antigenicity, and polyethylene Other known effects of alcohol on therapeutic proteins or analogs). The polyethylene glycol molecule (or other chemical moiety) must be attached to the protein, taking into account the effect on the functional or antigenic domain of the protein. There are many subsequent methods available to those skilled in the art, such as described in EP 0401 384, which is incorporated herein by reference (coupling PEG to G-CSF), see also Malik et al. -28- Standards apply to Chinese National Standards (CNS) 丨 Gx297 (Please read the notes on the back before filling out this page),? Τ Printed by the Consumers' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 1221482 A7 B7 V. Description of Invention (26)

Exp· Hematol·也：1028_1035(1992)(其中報導使用 tresyl chloride進行CM-CSF的聚乙二醇化）。例如，可經由反應性基’如自由胺基或羧基透過胺基酸殘基共價地結合的聚乙二醇。反應性基的可讓活化聚乙二醇分子結合者。具有自由胺基的安基酸殘基可包括離胺酸殘基和N-端胺基酸殘基；而具有自由羧基者可包括天冬胺酸殘基，穀胺酸殘基和C-端胺基酸殘基。氫硫基也可以作爲接著聚乙二醇分子所用的反應性基。對於治療目的較佳者爲在胺基上的接著，例如在N -端或離胺酸胺基上的接著。此外，可能有特別需要經N-端化學修飾的蛋白質。使用聚乙一醇作爲本發明組合物的闡示例子時，可以選用各種聚乙二醇分子（以分子量，分枝，等來分），反應混合物中聚乙二醇分子對蛋白質（或肽）分子的比例，要進行的聚乙二醇化反應之類別，及得到所選N -端聚乙二醇化蛋白質之方法。取传N -端聚乙二醇化製備物的方法（亦即，必要時將此邵份體從其他單聚乙二醇化部份體分離出）可爲從一族聚乙二醇化蛋白質分子純化出N-端聚乙二醇化物質。選擇性 N-端化學修飾可經由還原性烷基化來完成，其係利用在特別蛋白質中可用來衍化的不同類別第一胺基（離胺酸相對於 N-端）所具之差別反應性。在恰當反應條件下，可以達到用含羰基聚合物在贝_端進行之實質選擇性蛋白質衍化。合成OPG二體物可以經由各種化學交聯程序來製備成。 OPG單體可以經由可保留或增強0pG生物活性之任何方式予以化學聯結。有各種化學交聯劑可以依所欲蛋白質二體物的 -29- 本紙張尺度適用中國國家標準（CNS )八4規格（21()x297公羡) '' --- (請先閲讀背面之注意事項再填寫本頁)Exp. Hematol. Also: 1028_1035 (1992) (where tresyl chloride was reported for PEGylation of CM-CSF). For example, polyethylene glycol may be covalently bonded through an amino acid residue via a reactive group ' such as a free amine group or a carboxyl group. Reactive groups allow the binding of polyethylene glycol molecules to be activated. Amino acid residues with free amino groups may include lysine residues and N-terminal amino acid residues; while those with free carboxyl groups may include aspartic acid residues, glutamic acid residues, and C-terminal Amino acid residues. A hydrogen sulfide group can also be used as a reactive group for adhering a polyethylene glycol molecule. Preferred for therapeutic purposes is the attachment to an amine group, such as the N-terminus or an amine group. In addition, there may be proteins that specifically require N-terminal chemical modification. When polyethylene glycol is used as an illustrative example of the composition of the present invention, various polyethylene glycol molecules (divided by molecular weight, branching, etc.) can be selected. The polyethylene glycol molecules in the reaction mixture are compared to the protein (or peptide) molecules. The ratio, the type of pegylation reaction to be performed, and the method to obtain the selected N-terminal pegylated protein. The method of extracting N-terminal PEGylated preparations (ie, isolating this fraction from other mono-pegylated moieties if necessary) can purify N from a family of PEGylated protein molecules -Terminal pegylated material. Selective N-terminal chemical modification can be accomplished through reductive alkylation, which takes advantage of the differential reactivity of different classes of first amine groups (relative amino acids with respect to the N-terminus) that can be used for derivatization in specific proteins . Under proper reaction conditions, a substantially selective protein derivatization with a carbonyl-containing polymer at the shell end can be achieved. Synthetic OPG dimers can be prepared via various chemical crosslinking procedures. OPG monomers can be chemically linked by any means that can retain or enhance the biological activity of OpG. There are various chemical cross-linking agents that can be used according to the desired protein dimer. -29- This paper size applies to China National Standard (CNS) 8-4 specifications (21 () x297 public envy) '' --- (Please read the back (Please fill in this page again)

、1T Λ 1221482 A7 五、發明説明（27 ) 性質而選用。例如，交聯劑可爲短且相當剛性者或爲較長而更柔靭性者，可爲生物可逆者，及可提供減低的免疫遺傳性或較長的藥物動力學半生期者。經濟部中央標準局員工消費合作社印製 ^-- (請先閱讀背面之注意事項再填寫本頁) 於一實施例中，0PG分子係經由胺基端以兩步驟合成而鍵結在一起（參看實施例12)。於第一步驟中，〇PG係經在胺基端化學改質以導入經保護的硫醇，其在純化後予以去保護並用爲一接著點供透過各種交聯劑與一第二OPG分子進行部位-特定性拼合所用。胺基端交聯包括，但不限於，雙硫鍵，使用短鏈，雙官能性脂族交聯劑的硫醚鍵聯，及對各種長度’雙官能聚乙二醇交聯劑的硫醚鍵聯（蛋白質，，啞铃 ··)。此外，OPG二體物的PEG啞鈐合成也涵蓋彼等合成的副產物，稱爲”單鈴"（monobell)者。〇PG單鈴包括偶合到一線型雙έ旎PEG的單體而具有一自由聚合物端者。另外，〇pG 也可以直接透過各種胺特定性同雙官能***聯技術而交聯’其包括使用藥劑例如：二伸乙基三胺五乙酸二酐 (DTPA)，對·苯幷輥（pBQ)或辛二酸二（磺酸基丁二醯亞胺）鹽（B S )以及技藝中熟知的其他藥劑。也可以直接用藥劑例如亞胺基硫烷（iminothi〇lane)在各種雙官能，硫醇特定*** 聯劑，例如PEG雙順丁烯二醯亞胺存在中將〇PG硫醇化並在一步驟方法中達到二體化及/或啞鈐。另外也包括從天然來源及從經轉染宿主細胞純化〇PG之方法。该純化方法可採用一或多種標準蛋白質純化步驟以恰當順序進行而得到經純化的蛋白質。其層析術步驟可包括離子交換，膠濾，疏水性作用，逆相，層析焦聚 -30- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公餐） 1221482 經濟部中央標準局員工消費合作社印製 A7 _B7__ 五、發明説明（28 ) (chromatofocusing)，採用抗-OPG抗體或生物素-鏈抗生物素蛋白親和性複合物的親和性層析術等。抗體本發明也涵蓋可特定地結合到OPG的抗體。抗體產生所用的抗原可爲全長度多肽或跨越一部份OPG序列的肽。產生對 OPG具反應性的多株或單株抗體所用之免疫學程序係諳於此技者所知悉的（參看，例如，Harlow and Lane，Antibodies : A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor Ν·Υ· (1988))。如此所產生的抗體經使用標準的酵素聯結免疫吸著檢定鑑定其結合特定性和抗原決定部位辨識作用。抗體也包括嵌合型抗體其具有衍生自不同物種的可變和固定域區。於一實施例中，該嵌合型抗體爲具有鼠類的可變域和人類固定域之人化抗體。此外也涵蓋接枝到人類架構的互補決定區（所謂的CDR·接枝抗體）。嵌合型和CDR·接枝抗體係經由諳於此技者所知悉的重組方法製成的。另外也涵蓋在小鼠體内製成的人類抗體。本發明抗-OPG抗體可用爲親和劑以從生物樣品中純化 OPG(參看實施例1 0)。於一方法中，係將抗體固定化在 CnBr活化Sepharose上並用抗體-Sepharose拼合物管柱從液體樣品中取出OPG。抗體也可作爲診斷劑而於下述方法中用來偵檢和分量分析生物樣品中的OPG。醫藥組合物本發明也提出醫藥組合物其包括治療有效量的本發明多肽及醫藥可接受的稀釋劑，載劑，溶解化劑，乳化劑，防腐 -31 - 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁) .泰· 、τ 1221482 A7 B7 五、發明説明（29 ) 劑及/或佐劑。"治療有效量”一詞意指針對指定的病況和給藥途徑可提供治療效用的量。該組合物可爲液體或冷凍乾燥形式且包括稀釋劑（Tris，乙酸鹽或磷酸鹽緩衝劑）其可具各種pH値和離子強度；溶解化劑例如Tween或Polysorbate ; 載劑例如人類血清白蛋白或明膠；防腐劑例如硫柳汞或芊醇；及抗氧化劑例如抗壞血酸或偏亞硫酸氫鋼。此外也涵蓋包括用水溶性聚合物改質過以增加溶解性或安定性的OPG 之組合物。組合物也可包括將OPG摻加到脂質體，微乳液，微膠粒或囊泡中以控制時間内的輸送者。特別者，OPG組合物可包括摻加到聚合物基質例如水凝膠，矽酮，聚乙烯，乙烯-乙酸乙烯酯共聚物，或生物可分解的聚合物之内者。水凝膠的例子包括聚甲基丙烯酸羥烷基酯（p-HEMA)，聚丙烯醯胺，聚甲基丙烯醯胺，聚乙烯基吡咯啶酮，聚乙烯醇和各種聚電解質複合物。生物可分解性聚合物的例子包括聚乳酸（PLA)，聚乙醇酸（PGA)，PLA和PGA共聚物，聚醯胺和聚醯胺/聚酯共聚物。其他控制釋放方劑包括微膠囊劑，微球粒，巨分子複合物和可經由注射給藥的聚合物珠粒。經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 特定組合物的選擇決定於許多因素，包括正治療的病況，給藥途徑和所欲藥物動力學參數。適合於醫藥組合物的成份之更詳盡縱覽載於 Remington’s Pharmaceutical Sciences·第 18 版，A. R. Gennaro，ed. Mack，Easton，PA (1980) 0 本發明組合物可經由注射給藥，如皮下，靜脈内或肌肉内，或經由口服，鼻，肺或直腸給藥。最後給藥途徑係依許多因素而選定且可由諳於此技者予以確定。 -32- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（30 ) ' 一' 本發明也提出醫藥組合物，其包括治療有效量的本發明柱酸加上醫藥可接受的佐劑。核酸組合物適合用來將部份或全部的QPG編碼區輸送給細胞和組織作爲反意或基因治療法的一部份。治療方法骨組織係提供對身體的支持且其包括礦物質（大部份爲鈣和磷），膠蛋白性和非膠蛋白性蛋白質基質，及細胞。骨中有三類細胞，亦即骨母細胞，成骨細胞和蝕骨細胞，參與骨連續形成和吸除的動態程序中。成骨細胞可促進骨組織的形成而蚀骨細胞則與吸除作用有關。吸除，或骨基質和礦物質的溶解，相對於骨形成爲一快速且有效率的程序且可從骨釋出大量的礦物質。蝕骨細胞係參與骨骼組織正常再塑的調節及參與激素所謗導的吸除作用。例如，對應於細胞外液#5離子濃度減低而分泌的副甲狀腺激素即可能刺激吸除作用。相反地，吸除作用的抑制爲降血鈣素的主要功把。此外’維生素D的代謝物會變更骨對副甲狀腺激素和降血1¾素的回應性。於骨骼成熟後，骨骼中的骨含量即反映出骨形成和骨吸除的平衡（或不平衡）。尖峯骨質量係發生於骨骼成熟後四十歲之前。於四十歲與五十歲之間，平衡會偏移而由骨吸除作用主導。骨質量隨年齡增加的不可避免減少在女性會比男性起始得較早且在某些女性停經後更明顯加速（主要爲高加索系和亞洲系者）。貧骨症爲概括地有關任何種骨質減少到低於正常水平之病 -33 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210父297公釐7 (請先閲讀背面之注意事項再填寫本頁) .裝· 訂 -嫩- 1221482 A7 B7 五、發明説明（31 ) 況。彼等病況可能起源於骨合成速率的減低或骨破壞速率的增加或兩者。最普遍的貧骨症形式爲原發性骨稀鬆症，也稱爲斷經後和老年骨稀鬆症。此種形式的骨稀鬆症爲全面性骨質隨年齡損失之結果且通常爲在正常骨形^速率^ 下骨吸除作用增加之結果。在美國全部白人女性中約有2 $ 至30A發展出系統性骨稀鬆症。在45歲和更老的女人中，骨稀鬆症與髖，股，頸和粗隆間骨折之間存在著直接相關性。老男人係在5 0至7 0歲之間發展出系統性骨稀鬆症，不過此症主要影響到女性。、斷經後和老年骨稀鬆症的肇因尚未得知。有幾項因素係經鑑定可能促成該病況者。其中包括激素水平隨年齡的變’I 及因小腸對鈣和其他礦物質的吸少減少所致鈣消耗不足。其治療通常包括激素治療或食物補充以期延緩該程序。不過，到目前爲止，尚未存在對骨損失的有效治療法。本發明提出一種使用治療有效量的〇pG治療骨病的方法。骨病可爲具有淨骨質損失特徵之任何病（貧骨症或骨質溶解）。一般而言，在有需要壓抑骨吸除速率時即預期用〇ρσ 治療。依此，該治療可用來在吸除速率高於正常値時減低經濟部中央標準局員工消費合作社印製，赛-- (請先閲讀背面之注意事項再填寫本頁) 骨吸除速率或將骨吸除減少到低於正常水平以彌補低於正常水平的骨形成。 OPG可治療的病況包括下列：骨稀趁症’例如原發性骨稀鬆症，内分泌骨稀鬆症（甲狀腺官能過旺症’副甲狀腺官能過旺症，克興氏徵候群，和肢端肥大病），遣傳性和先性形式的骨稀鬆症（成f不全， -34 木紙張尺度適用中國國家標準（CNS ) A4規 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（泣）同胱胺酸尿症，Menkes，徵候群，和Riley-Day徵候群）及因肢固定所致骨稀鬆症。相哲德氏骨病（變形性骨炎），成人和年青人。骨髓炎，或骨中感染性傷害導致骨損失。固體腫瘤（胸，肺和腎）和血液學惡症（多發性骨髓瘤，淋巴瘤和白血病）所致高躬血症，自發性高舞血症，及與甲狀腺官能過旺症和腎官功相關聯的高鈣血症。貧骨症，於手術後者，類固醇服用所謗導者，及與小腸和大腸疾病與慢性肝病和腎病相關聯者。骨壞死，或骨細胞死亡，與創傷性傷害相關聯者或與高歇氏病，鐮狀細胞性貧血，系統性紅斑狼瘡和其他病況相關聯的非創傷性壞死。因風濕性關節炎所致骨質流失。齒骨膜骨質流失。溶骨性轉移。要了解者，OPG可以單獨使用或與其他因子配合使用來治療骨病。於一實施例中，係用促骨堆積素配合治療有效量的可刺激骨形成之因子。彼等因子包括但不限於從BMP-1命名到BMP-12的骨形態發育因子，轉形生長因子(TGF-/3) 和TGF·卢族成員，間白素-1抑制劑，TNF從抑制劑，副甲狀腺激素和其類似物，副甲狀腺相關性蛋白質和其類似物，E 系***素，雙膦酸鹽（例如alendronate和其他），及骨強化性礦物質例如氟化物和鈣。下列的實施例係提供來更完整地闡述本發明，但不可視爲 35- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） T 口 (請先閱讀背面之注意事項再填寫本頁) 1221482 A7 B7 五、發明説明（33 ) 係要限制其範園者。實施例1 老鼠OPG cDNA的鑑定和分離 cDNA選殖和分析所用物質和方法皆載於Maniatis et aH 述之中。聚合酶連鎖反應（PCR)係以Perkin-Elmer 9600熱循環器用製造商規定的PCR反應混合物（Boehringer-Mannheim) 和引子濃度實施的。通常，25-50微升的反應係在94°C變性，接著20_40次的循環：94Ό 5秒鐘，50_6(TC 5秒鐘，和72 °C 3-5分鐘。接著在72 °C處理反應物3_5分鐘。然後以 Maniatis et al.，同上中所述凝膠電泳法分析反應物。經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 使用得自胎d20腸的mRNA構成cDNA庫供EST分析用 (Adams et al. Science 252· 1651-1656(1991))。切出老鼠的胚胎，取出整個多育中的小腸和大腸並在PBS中洗清。經由酸性硫氰酸胍-驗氣仿萃取純化整個細胞RNA(Chomczynski and Sacchi Anal. Biochem. 162· 156-159，（1987))。用該整個 RNA製備物經由使用製造商的建議程序吸附到Dynabeads Oligo (dT)25 (Dynal Corp)及從其洗提出而得聚（A+) mRNA 部份。使用 Superscript Plasmid System (Gibco BRL， Gaithersburg，Md)製備經無規引導的cDNA庫。使用含有内 Not I限制部位的無規cDNA引子來起始第一股的合成且其具有下示序列： 5,-AAAGGAAGGAAAAAAGCGGCCGCTACANNNNNNNNT-3, (SEQIDNO ： 1)1T Λ 1221482 A7 V. Description of the invention (27) The nature is selected. For example, the cross-linking agent may be short and fairly rigid or longer and more flexible, may be bioreversible, and may provide reduced immune genetics or longer pharmacokinetic half-life. Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs ^ (Please read the notes on the back before filling this page) In one embodiment, the 0PG molecule is synthesized in two steps via the amine end and bonded together (see Example 12). In the first step, OPG is chemically modified at the amine end to introduce a protected thiol, which is deprotected after purification and used as a point-to-point for the passage through various cross-linking agents and a second OPG molecule. Site-specificity. Amine-terminated cross-linking includes, but is not limited to, disulfide bonds, thioether linkages using short-chain, bifunctional aliphatic crosslinkers, and thioethers for various lengths of 'bifunctional polyethylene glycol crosslinkers. Linking (protein, dumbbell ...). In addition, the PEG dumb synthesis of OPG dimers also covers the by-products of their synthesis, known as "monobells." PG monobells include monomers that are coupled to first-line double-stranded PEG and have A free polymer end. In addition, OpG can also be directly cross-linked through various amine-specific and bifunctional cross-linking techniques. This includes the use of agents such as: diethylene triamine pentaacetic dianhydride (DTPA). · Phenylhydrazone (pBQ) or bis (sulfosuccinimide) salt (BS) and other agents well known in the art. Agents such as iminothioane can also be used directly OPG is thiolated in the presence of various bifunctional, thiol-specific cross-linking agents, such as PEG dicis-butene difluorenimide, and dimerization and / or dumbness is achieved in a one-step process. Also included are natural Source and method of purifying OPG from transfected host cells. This purification method can use one or more standard protein purification steps in the proper order to obtain purified protein. Its chromatography steps can include ion exchange, gel filtration, Hydrophobic effect, reverse phase,焦焦聚 -30- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 public meal) 1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 _B7__ V. Invention Description (28) (chromatofocusing) OPG antibody or biotin-chain avidin affinity complex affinity chromatography, etc. Antibodies The present invention also encompasses antibodies that can specifically bind to OPG. Antigens used for antibody production can be full-length polypeptides or span a Partial OPG sequence peptides. The immunological procedures used to produce multiple or monoclonal antibodies that are reactive to OPG are known to those skilled in the art (see, eg, Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. (1988)). The antibodies thus produced were identified for their binding specificity and epitope recognition using standard enzyme-linked immunosorbent assays. Antibodies also include chimeric antibodies It has variable and fixed domain regions derived from different species. In one embodiment, the chimeric antibody is a murine variable domain and Humanized antibodies of the fixed domain type. In addition, it also covers the complementarity-determining regions grafted into the human framework (so-called CDR · grafted antibodies). Chimeric and CDR · grafted anti-systems are recombined by those skilled in the art It is made by the method. It also covers human antibodies made in mice. The anti-OPG antibody of the present invention can be used as an affinity agent to purify OPG from a biological sample (see Example 10). In one method, The antibody was immobilized on CnBr-activated Sepharose and OPG was removed from the liquid sample using an antibody-Sepharose conjugate column. Antibodies can also be used as diagnostic agents in the methods described below to detect and fractionate OPG in biological samples. Pharmaceutical composition The present invention also proposes a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of the present invention and a pharmaceutically acceptable diluent, carrier, dissolving agent, emulsifier, antiseptic -31-This paper is in accordance with Chinese national standards (CNS ) Α4 size (210X 297 mm) (Please read the notes on the back before filling out this page). ··· τ 1221482 A7 B7 V. Description of the invention (29) Agent and / or adjuvant. The term "therapeutically effective amount" means an amount that provides therapeutic utility for a given condition and route of administration. The composition may be in liquid or lyophilized form and includes a diluent (Tris, acetate or phosphate buffer) It can have various pHs and ionic strengths; solubilizing agents such as Tween or Polysorbate; carriers such as human serum albumin or gelatin; preservatives such as thimerosal or methanol; and antioxidants such as ascorbic acid or metabisulfite steel. In addition also Compositions that include OPG modified with water-soluble polymers to increase solubility or stability are also encompassed. Compositions can also include the incorporation of OPG into liposomes, microemulsions, micelles, or vesicles to control time Conveyor. In particular, the OPG composition may include those incorporated into a polymer matrix such as a hydrogel, silicone, polyethylene, an ethylene-vinyl acetate copolymer, or a biodegradable polymer. Hydrogel Examples include polyhydroxyalkyl methacrylate (p-HEMA), polyacrylamide, polymethacrylamide, polyvinylpyrrolidone, polyvinyl alcohol and various polyelectrolyte compounds Examples of biodegradable polymers include polylactic acid (PLA), polyglycolic acid (PGA), PLA and PGA copolymers, polyamides and polyamide / polyester copolymers. Other controlled release formulas include microcapsules , Microspheres, macromolecular complexes, and polymer beads that can be administered by injection. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). Selection of specific composition Many factors, including the condition being treated, the route of administration, and the desired pharmacokinetic parameters. A more detailed overview of the ingredients suitable for pharmaceutical compositions is contained in Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed. Mack, Easton PA (1980) 0 The composition of the present invention can be administered by injection, such as subcutaneously, intravenously or intramuscularly, or by oral, nasal, pulmonary or rectal administration. The final route of administration is selected based on many factors and can be selected from 谙Determined by this technician. -32- This paper size is applicable to China National Standard (CNS) A4 (210X 297 mm) 1221482 Employees' cooperation with the Central Standards Bureau of the Ministry of Economic Affairs Print A7 B7 5. Description of the invention (30) 'a' The present invention also proposes a pharmaceutical composition, which includes a therapeutically effective amount of the column acid of the present invention plus a pharmaceutically acceptable adjuvant. The nucleic acid composition is suitable for Or the entire QPG coding region is delivered to cells and tissues as part of antipathy or gene therapy methods. Therapeutic methods The bone tissue system provides support to the body and includes minerals (mostly calcium and phosphorus), gelatin Sexual and non-gliaminous protein matrices, and cells. There are three types of cells in bone, namely osteoblasts, osteoblasts and osteoclasts, which participate in the dynamic process of continuous bone formation and aspiration. Osteoblasts can promote the formation of bone tissue, while osteoblasts are associated with aspiration. Aspiration, or dissolution of bone matrix and minerals, is a fast and efficient procedure with respect to bone formation and can release a large amount of minerals from bone. The osteoclast cell line is involved in the regulation of normal remodeling of skeletal tissues and in the absorption effect mediated by hormones. For example, parathyroid hormone secreted in response to a decrease in the concentration of extracellular fluid # 5 may stimulate aspirating effects. Conversely, the suppression of absorption is the main function of calcitonin. In addition, a 'vitamin D metabolite changes bone's responsiveness to parathyroid hormones and hypotonin. After the bone matures, the bone content in the bone reflects the balance (or imbalance) of bone formation and bone resorption. Spike bone mass occurs before forty years of age after bone maturation. Between the ages of forty and fifty, balance shifts and is dominated by bone aspiration. The unavoidable decrease in bone mass with age will start earlier in men than men and will accelerate significantly after menopause in some women (mainly Caucasian and Asian). Osteoporosis is a disease that generally relates to any type of bone that is reduced to below normal levels. -33-This paper size applies Chinese National Standard (CNS) A4 specifications (210 father 297 mm 7 (Please read the precautions on the back before filling out (This page). Binding · Binding-Twelve-1221482 A7 B7 V. Description of the invention (31). Their conditions may originate from a decrease in the rate of bone synthesis or an increase in the rate of bone destruction or both. The most common form of anemia Primary osteoporosis, also known as post-menopausal and senile osteoporosis. This form of osteoporosis is the result of a general loss of bone with age and is usually a bone resorption at a normal bone shape ^ rate ^ As a result of increased effects, systemic osteoporosis develops in approximately 2 to 30 A of all white women in the United States. In women 45 and older, osteoporosis is associated with hip, femoral, neck, and intertrochanteric fractures. There is a direct correlation between them. Old men develop systemic osteoporosis between the ages of 50 and 70, but the disease mainly affects women. The cause of postmenopausal and osteoporosis in the elderly is unknown. . Several factors have been identified that may contribute Patients with conditions. These include changes in hormone levels with age, and inadequate calcium consumption due to reduced intake of calcium and other minerals in the small intestine. Treatment usually includes hormone therapy or food supplementation to delay the procedure. However, until now So far, there has not been an effective treatment for bone loss. The present invention proposes a method for treating bone disease using a therapeutically effective amount of oPG. Bone disease may be any disease (osteoporosis or osteolysis) with characteristics of net bone loss. Generally speaking, when there is a need to suppress the rate of bone resorption, it is expected to be treated with 0ρσ. Based on this, the treatment can be used to reduce the print by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs when the absorption rate is higher than normal. -(Please read the precautions on the back before filling out this page) Bone removal rate or reduce bone removal to below normal levels to compensate for bone formation below normal levels. OPG treatable conditions include the following: 'Such as primary osteoporosis, endocrine osteoporosis (hyperthyroidism', parathyroidism, Kerry's syndrome, and acromegaly Disease), degenerative and pro-formal forms of osteoporosis (incomplete, -34 wood paper standard applicable to Chinese National Standards (CNS) A4 regulations 1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (Crying) homocystinuria, Menkes, syndrome, and Riley-Day syndrome) and osteoporosis due to limb immobilization. Sage De's bone disease (deformative osteitis), adults and young people. Osteomyelitis, or infectious injury to the bone, leads to bone loss. Hypertensive hememia due to solid tumors (thorax, lungs, and kidneys) and hematological malignancies (multiple myeloma, lymphoma, and leukemia), spontaneous hypertensive Disease, and hypercalcemia associated with hyperthyroidism and renal function. Bone anemia, the latter after surgery, steroid administration, and those associated with small and large bowel disease with chronic liver and kidney disease . Osteonecrosis, or bone cell death, nontraumatic necrosis associated with traumatic injury or Gaucher's disease, sickle cell anemia, systemic lupus erythematosus, and other conditions. Bone loss due to rheumatoid arthritis. Periosteum bone loss. Osteolytic metastasis. To understand, OPG can be used alone or in combination with other factors to treat bone disease. In one embodiment, an osteotropin is used in combination with a therapeutically effective amount of a factor that stimulates bone formation. These factors include, but are not limited to, bone morphogenesis factors named from BMP-1 to BMP-12, transforming growth factor (TGF- / 3) and TGF · Lu family members, melanin-1 inhibitors, and TNF from inhibition Agents, parathyroid hormones and their analogs, parathyroid-related proteins and their analogs, E-type prostaglandins, bisphosphonates (such as alendronate and others), and bone-reinforcing minerals such as fluoride and calcium. The following examples are provided to illustrate the present invention more completely, but should not be regarded as 35- This paper size is applicable to Chinese National Standard (CNS) A4 specifications (210X297 mm) T-mouth (Please read the precautions on the back before filling this page ) 1221482 A7 B7 V. The description of invention (33) is for those who want to restrict their fields. Example 1 Identification and isolation of mouse OPG cDNA The materials and methods used for cDNA colonization and analysis are described in Maniatis et aH. The polymerase chain reaction (PCR) was performed using a Perkin-Elmer 9600 thermal cycler with a PCR reaction mixture (Boehringer-Mannheim) and primer concentrations specified by the manufacturer. Usually, 25-50 μl of the reaction is denatured at 94 ° C, followed by 20-40 cycles: 94Ό 5 seconds, 50_6 (TC 5 seconds, and 72 ° C 3-5 minutes. The reaction is then treated at 72 ° C 3-5 minutes. Then analyze the reactants by gel electrophoresis as described in Maniatis et al., As described above. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). The d20 intestinal mRNA constitutes a cDNA library for EST analysis (Adams et al. Science 252 · 1651-1656 (1991)). The mouse embryos were excised, the whole small intestine and large intestine were removed and washed in PBS. Acidic guanidinium thiocyanate-gas-mimetic extraction purification of whole cell RNA (Chomczynski and Sacchi Anal. Biochem. 162 · 156-159, (1987)). This whole RNA preparation was adsorbed to Dynabeads Oligo using the manufacturer's recommended procedure (dT) 25 (Dynal Corp) and poly (A +) mRNA fractions eluted from it. Superscript Plasmid System (Gibco BRL, Gaithersburg, Md) was used to prepare a randomly-guided cDNA library. Uses containing internal Not I restriction Random cDNA primers And it has the sequence shown below: 5, -AAAGGAAGGAAAAAAGCGGCCGCTACANNNNNNNNT-3, (SEQIDNO: 1)

Not I 對於第一股合成要組合三個分開的反應，其中含有2.5微 -36 - 本紙張尺度適用中國國家榡準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局貝工消費合作社印製 A7 _ B7__ 五、發明説明（34 ) 克的poly(A) RNA和120毫微克，360毫微克或1，080毫微克的無規引子。於第二股合成之後，用酚：氣仿：異戊醇（25 : 24 : 1比例）混合物分別萃取該等反應產物且接著予以乙醇沈澱。將三個反應的雙股（ds) cDNA產物合併並連接到下列ds 寡核苷酸轉接子（adapter): 5'-TCGACCCACGCGTCCG-3· (SEQ ED NO : 2) S'-GGGTGCGCAGGCp-S' (SEQ ID NO ： 3) 連接後，用Not I將cDNA消化到完全，再用酚：氣仿：異戊醇（25 : 24 : 1)萃取及乙醇沈澱。然後將再懸浮的cDNA用裝設著 Superscript Plasmid System (Gibco BRL，Gaithersburg， Md)的預製管柱依製造商所建議者進行凝膠過濾予以尺寸分級。將含有最大cDNA產物的兩洗提份合併，乙醇沈澱再導引地連接到經Not I和Sal I消化過的pMOB媒體 DNA(Strathmann et al, 1991)。連接好的 cDNA 即經由 electroporation導到勝任的ElectroMAX DH10B大腸桿菌中 (Gibco BRL，Gaithersburg, MD)。對於自動序列分析，係將約10,000轉形體置於裝著氨芊黴素補充LB營養培養基的20 公分X20分分瓊脂板上平板培養。將產生的菌落挑起並列佈在裝著200毫升L-肉湯，7.5%甘油，和50微克/毫升氨芊黴素的9 6洞微滴板上。將培養物置於37°C下生長整個晚上，使用無菌9 6栓複製工具製成重複的微滴板組，然後將兩組都貯存在-80°C供後續分析所用。對於全長度cDNA選殖，係將約一百萬轉形體置於各含約1〇,〇〇〇菌落的96細菌氨t黴彖板上進行平板培養。將各池的質體DNA用Qiagen •37- 本紙張又度適用中國國家標準（CNS ) Α4規格（21〇Χ297公釐） ----------0------、τ------^ (請先閲讀背面之注意事項再填寫本頁) 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（35 )Not I has to combine three separate reactions for the first synthesis, which contains 2.5 micro-36-this paper size applies to China National Standards (CNS) A4 specifications (210X297 mm) 1221482 Shellfish Consumer Cooperative, Central Standards Bureau, Ministry of Economic Affairs A7 _ B7__ printed 5. Description of the invention (34) grams of poly (A) RNA and 120 nanograms, 360 nanograms, or 1,080 nanograms of random primers. After the second synthesis, the reaction products were separately extracted with a mixture of phenol: aeroform: isoamyl alcohol (25: 24: 1 ratio) and then ethanol precipitated. The three reacted double-stranded (ds) cDNA products were combined and ligated to the following ds oligonucleotide adapters: 5'-TCGACCCACGCGTCCG-3 · (SEQ ED NO: 2) S'-GGGTGCGCAGGCp-S ' (SEQ ID NO: 3) After ligation, the cDNA was digested to completion with Not I, and then extracted with phenol: aeroform: isoamyl alcohol (25: 24: 1) and ethanol precipitation. The resuspended cDNA was then graded by gel filtration using a preformed column equipped with a Superscript Plasmid System (Gibco BRL, Gaithersburg, Md) according to the manufacturer's recommendations. The two eluates containing the largest cDNA product were combined and ethanol-precipitated and then ligated to the pMOB media DNA digested with Not I and Sal I (Strathmann et al, 1991). The ligated cDNA was electroporated into competent ElectroMAX DH10B E. coli (Gibco BRL, Gaithersburg, MD). For automatic sequence analysis, approximately 10,000 transformants were plated on a 20 cm x 20 cent agar plate filled with ampicillin-supplemented LB nutrient medium. The resulting colonies were picked up and arranged side by side on a 96-well microtiter plate filled with 200 ml of L-broth, 7.5% glycerol, and 50 µg / ml ampicillin. Cultures were grown overnight at 37 ° C. Repeated microtiter plate sets were made using a sterile 96-pin replication tool, and both sets were stored at -80 ° C for subsequent analysis. For full-length cDNA colonies, about one million transformants were plated on 96 bacterial ammonia bacterial mold plates each containing about 10,000 colonies. Use Qiagen • 37- plastid DNA from each cell. This paper is again applicable to China National Standard (CNS) A4 specification (21〇 × 297 mm) ---------- 0 ------ 、 τ ------ ^ (Please read the notes on the back before filling out this page) 1221482 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of Invention (35)

Plasmid Maxi Kit (Qiagen Corp·，Germany)分別地離析出來並佈列在9 6洞微滴板中供PCR分析所用。要定序無規胎老鼠腸cDNA株時，係將甘油儲料解凍，並取小液份以1 : 25稀释到蒸餾水中。於含有下列寡核：g：酸的 PCR反應混合物（Boehringer_Mannheim)中加入約3.0微升的稀細菌培養液： 5,-TGTAAAACGACGGCCAGT-3, (SEQIDNO ： 4) 5'-CAGGAAACAGCTATGACC-3' (SEQIDNO ： 5) 將反應物置於熱循環器（Perkin-Elmer 9600)中用下列循環條件下培育：94°C 2分鐘；3 0個循環的：94°C 5秒鐘，50°C 5秒鐘，和72°C 3分鐘；接著72°C 4分鐘。在熱循環器中培育之後，用2.0毫升水稀釋反應物。擴增後的DNA片段再用 Centricon管柱（Princeton Separations)以製造商建議的程序進一步純化。將PCR反應產物置於Applied Biosystems 373A自動DNA定序儀上用T3引子（寡核甞酸353-23 ; 5·-CAATTAACCCTCACTAAAGG-3丨)（SEQ ID NO : 6) Taq染料-終止區反應（Applied Biosystems)遵照製造商的建議程序進行序列分析。從無規挑取cDNA株得到之5 ’核甞酸序列經轉譯後，與既有的已知蛋白質序列數據庫以修改過的FASTA程式版本進行比對（Pearson et al· Meth· Enzymol. 183· (1990))。此外也對轉譯序列分析其是否含有在所有已知的腫瘤壞死因子受體（TNFR)超族成員中都發現過的特定富含半胱胺酸蛋白質樣式（Smith et al· Cell 丛，959-962 (1994))，其中使用經 -38- 本紙張尺度適用中國國家標準（CNS ) Μ規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（36 )Plasmid Maxi Kit (Qiagen Corp., Germany) was isolated and arranged in 96-well microtiter plates for PCR analysis. To sequence the intestinal cDNA strain of a random fetus, the glycerol stock is thawed and a small solution is diluted 1:25 into distilled water. To a PCR reaction mixture (Boehringer_Mannheim) containing the following oligo: g: acid, about 3.0 microliters of a dilute bacterial culture broth was added: 5, -TGTAAAACGACGGCCAGT-3, (SEQIDNO: 4) 5'-CAGGAAACAGCTATGACC-3 '(SEQIDNO: 5) Place the reaction in a thermal cycler (Perkin-Elmer 9600) and incubate under the following cycling conditions: 94 ° C for 2 minutes; 30 cycles: 94 ° C for 5 seconds, 50 ° C for 5 seconds, and 72 ° C for 3 minutes; then 72 ° C for 4 minutes. After incubation in a thermal cycler, the reaction was diluted with 2.0 ml of water. The amplified DNA fragments were further purified using Centricon columns (Princeton Separations) using procedures recommended by the manufacturer. The PCR reaction product was placed on an Applied Biosystems 373A automatic DNA sequencer using T3 primers (oligonucleotide 353-23; 5 · -CAATTAACCCTCACTAAAGG-3 丨) (SEQ ID NO: 6) Taq dye-stop region reaction (Applied Biosystems) followed the manufacturer's recommended procedures for sequence analysis. The 5 'nucleotide sequence obtained from the randomly selected cDNA strain was translated and compared with an existing database of known protein sequences using a modified version of the FASTA program (Pearson et al. Meth. Enzymol. 183 · ( 1990)). In addition, the translation sequence was analyzed to see if it contained a specific cysteine-rich protein pattern found in all known members of the tumor necrosis factor receptor (TNFR) superfamily (Smith et al. Cell Plex, 959-962 (1994)), where the paper size is -38- This paper size applies to Chinese National Standards (CNS) M specifications (210X297 mm) (Please read the precautions on the back before filling this page) Order 1221482 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Printed by the cooperative A7 B7 V. Description of the invention (36)

Luethy et al.修改過（Protein Science 2_，139-146 (1994))的 Gribskov et al.之序列部面法（sequence profile method)(Proc. Natl. Acad· Sci· USA ϋ，4355-4359 (1987))。使用FASTA和剖面搜尋數據時，鑑別出一 EST，FRM(胎老鼠腸-1 )爲可能的新TNFR超族成員。FRI-1含有約600 bp ***體及約150胺基酸的LORF。數據庫中的最接近匹配爲人類第II型TNFR(TNFR-2)。比對過的區段顯示在TNFR-2和 FRI-1之間於此150胺基酸LORF有〜43%同系性。使用TNFR 超族的第一和第二兩富含半胱胺酸重複域進行剖面分析得到〜8的Z分，顯示該FRI-1基因可能編碼一新的族員。爲了推導出FRI-1產物的構造，乃篩選胎老鼠腸CDNA庫以期得到全長度株。下面的寡核甞酸係衍生自原始的FRI-1序列： 5,-GCATTATGACCCAGAAACCGGAC-3, (SEQIDNO ： 7) 5.-AGGTAGCGCCCTTCCTCACATTC-3 (SEQIDNO ： 8) 用這些引子於PCR反應中以篩選96池的質體DNA，各池含有來自10,000獨立CDNA株的質體DNA。將約1微克的質體池 DNA 置於 PCR反應混合物（Boehringer-Mannheim)中用 Perkin· Elmer 96洞熱循環器以下面的循環條件予以擴增：94Ό 2分鐘，1循環；94°C 15秒，然後65°C 45秒，30個循環；65°C 7 分鐘，1循環。之後以凝膠電泳分析PCR反應產物。96質體 DNA池中有i 3池產生具有預期相對分子量的擴增〇ΝΑ產物。使用來自一陽性池的DNA依上述來轉形勝任的Luethy et al. (Sequence profile method of Gribskov et al. (Protein Science 2_, 139-146 (1994))) (Proc. Natl. Acad · Sci · USA ϋ, 4355-4359 (1987 )). When using FASTA and profile search data, an EST, FRM (fetal rat intestine-1) was identified as a possible new TNFR superfamily member. FRI-1 contains about 600 bp of insert and about 150 amino acids of LORF. The closest match in the database was human type II TNFR (TNFR-2). The aligned segments show ~ 43% homology between the 150 amino acid LORF between TNFR-2 and FRI-1. Profile analysis using the first and second cysteine-rich repeat domains of the TNFR superfamily yielded a Z score of ~ 8, indicating that the FRI-1 gene may encode a new family member. In order to deduce the structure of the FRI-1 product, the fetal mouse intestinal CDNA library was screened to obtain a full-length strain. The following oligonucleotides are derived from the original FRI-1 sequence: 5, -GCATTATGACCCAGAAACCGGAC-3, (SEQIDNO: 7) 5.-AGGTAGCGCCCTTCCTCACATTC-3 (SEQIDNO: 8) Use these primers in a PCR reaction to screen 96 pools Each pool contains plastid DNA from 10,000 independent CDNA strains. Approximately 1 microgram of plastid pool DNA was placed in a PCR reaction mixture (Boehringer-Mannheim) and amplified using a Perkin · Elmer 96-hole thermal cycler under the following cycling conditions: 94Ό 2 minutes, 1 cycle; 94 ° C 15 seconds , Then 65 ° C for 45 seconds, 30 cycles; 65 ° C for 7 minutes, 1 cycle. The PCR reaction products were then analyzed by gel electrophoresis. The i 3 pool in the 96 plastid DNA pool produced amplified ONA products with expected relative molecular weights. Use DNA from a positive pool to transform a competent one as described above

ElectroMAX DH10B 大腸桿菌（Gibco BRL，Gaithersburg， _ -39- 本紙張尺度適用中國國家檩準（CNS ) A4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁) 、π 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（37 ) MD)。將約40,000轉形體接種在無菌硝基纖維素濾紙（BA-85, Schleicher and Schuell)上，然後用上面所得PCR產物的32p-dCTP標記物進行菌落雜合篩選。濾紙在5X SSC，50%去離子甲醯安，5X Denhardt's溶液。0.5% SDS，和100微克/毫升變性鮭魚***DNA之中在42°C預雜合2 - 4小時。然後將濾紙置於5X SSC，50%去離子甲醯安，2X Denhardfs溶液，0.1% SDS，100微克/毫升變性鮭魚***DNA，和〜5毫微克/毫升標記探測核酸之中在42°C下雜合〜18小時。之後將濾紙在2X SSC中室溫下洗10分鐘，IX SSC中55°C下洗10分鐘，及最後在0.5X SSC中5 5°C下洗10_15分鐘。於放射攝影後偵檢雜合菌落，然後再接種在硝基纖維素濾紙上進行二次篩選。在二次筛選下，分離出質體株（pBl.l)，再於含有100微克/ 毫升氨苄黴素的L-肉湯培養基中擴增而得到質體DNA。將該2.4kbpBl.l***體的兩股予以定序。用該pB 1.1***體序列進行公開數據庫的FASTA插尋以偵測任何既有的序列匹配及/或類似性。未發現到對任何已知基因或EST’s的匹配，雖則對人類和小鼠TNFR-2基因有45% 的相似性。在核甞酸序列的bp 124處發現一甲硫胺酸起始密碼，接著爲編碼401胺基酸殘基的LORF，其在bp 1327處終止。該401胺基酸殘基產物經預測在其N-端具有一由約31 殘基構成的疏水性信號肽，和4個潛在的N_鍵聯糖基化部位。使用PepPlot程式（Wisconsin GCG套裝軟體，8.1版）未鑑定出疏水性透膜跨越序列。然後用該推得之401胺基酸序列來插尋蛋白質數據庫。再度地，沒有既存的匹配，不過對 -40- 5^尺度適用中國國家標準（CNS ) A4規格（210X297公釐1 (請先閱讀背面之注意事項再填寫本頁) 、τ 1221482 A7 B7 五、發明説明（38 ) TNFR超族的許多成員，最顯著的是人類和小鼠TNFR- 2似乎有強相似性。用Pileup程式製備此新穎蛋白質與已知TNFR-超族成員的序列比對排列後，用PrettyPlot (Wisconsin GCG 套裝軟體，8.1版）修改。此排列顯示全長度FRI-1基因產物與所有其他TNFR族員之間有清楚的同系性。該等同系區的圖位係在TNFR族員的細胞外域，且對應於這些蛋白質所具配體結合域中所發現的四個富含半胱胺酸重複體中之三個。此結果可推測該FRI-1基因編碼·新穎的TNFR族員。由於沒有偵檢到透膜展區，因此我們預測其可能爲一分泌受體，類似於TNFR-1衍生的可溶性受體（〖〇1111〇61&1.?1：(^· Natl. Acad. Sci. USA ϋ 8331-8335 (1990))。由於該 FRI-1 基因所具的明顯生物活性（見下文），將該產物稱爲促骨堆積素（OPG) 〇實施例2 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁)ElectroMAX DH10B E. coli (Gibco BRL, Gaithersburg, _ -39- This paper size applies to China National Standard (CNS) A4 size (210X 297 mm) (Please read the precautions on the back before filling this page)) π 1221482 Economy Ministry of consumer cooperatives central Bureau of standards staff printed A7 B7 V. Description (37) MD) invention. Approximately 40,000 transformants were inoculated on sterile nitrocellulose filter paper (BA-85, Schleicher and Schuell), and then subjected to colony hybrid screening with the 32p-dCTP marker of the PCR product obtained above. Filter paper in 5X SSC, 50% deionized A safety acyl, 5X Denhardt's solution. In 0.5% SDS, and 100 g / ml denatured salmon sperm DNA at pre hybrid 42 ° C 2 - 4 hours. Filter paper was then placed in 5X SSC, 50% deionized metformin, 2X Denhardfs solution, 0.1% SDS, 100 μg / ml denatured salmon sperm DNA, and ~ 5 ng / ml labeled probe nucleic acid at 42 ° C Hybrid ~ 18 hours. The filters were then washed in 2X SSC at room temperature for 10 min, wash in IX SSC at 55 ° C 10 min, and a final wash in 0.5X SSC at 5 5 ° C 10_15 minutes. The hybrid colonies were detected after radiography, and then inoculated on nitrocellulose filter paper for secondary screening. Under the secondary screening, the plastid strain (pBl.l) was isolated and then amplified in an L-broth broth containing 100 μg / ml ampicillin to obtain plastid DNA. The two strands of the 2.4 kbpBl.l insert were sequenced. This pB 1.1 insert sequence was used for FASTA interpolation of public databases to detect any existing sequence matches and / or similarities. No match was found for any known gene or EST's, although there was a 45% similarity to human and mouse TNFR-2 genes. A methionine was found at bp 124 start at a password nuclear Chang acid sequence, followed by a LORF encoding 401 amino acid residues, which terminates at bp 1327 place. The 401 amino acid residue product is predicted to have a hydrophobic signal peptide consisting of about 31 residues at its N-terminus, and four potential N-linked glycosylation sites. Using the PepPlot program (Wisconsin GCG software package, version 8.1), no hydrophobic transmembrane spanning sequence was identified. The deduced 401 amino acid sequence was then used to interpolate the protein database. Again, there is no existing match, but the Chinese National Standard (CNS) A4 specification (210X297 mm1) is applied to the -40-5 ^ scale (please read the precautions on the back before filling this page), τ 1221482 A7 B7 V. Description of the Invention (38) Many members of the TNFR superfamily, most notably human and mouse TNFR-2 appear to have strong similarities. The novel protein was prepared using the Pileup program and aligned with known TNFR-superfamily members. , Modified with PrettyPlot (Wisconsin GCG software package, version 8.1). This arrangement shows a clear homology between the full-length FRI-1 gene product and all other members of the TNFR family. The map of this equivalent line is in the TNFR family Extracellular domain, and corresponds to three of the four cysteine-rich repeats found in the ligand-binding domains of these proteins. This result suggests that the FRI-1 gene encodes a novel TNFR family member Since no transmembrane zone was detected, we predict that it may be a secretory receptor, similar to the soluble receptor derived from TNFR-1 (〖〇1111〇61 & 1.? 1: (^ · Natl. Acad. Sci. USA ϋ 8331-8335 (1990)). Since The FRI-1 gene has obvious biological activity (see below), and the product is called osteotrophin (OPG). Example 2 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back first) (Fill in this page again)

組織中的OPG mRNA表現圖式用32P-dCTP標記的FRI-1 PCR產物作爲探測核酸進行複式人類組織北方氏吸潰（Clonetech)以偵檢人類轉錄體的尺寸及測定表現圖式。北方氏吸潰係在5X SSPE，50%甲醯胺，5X Denhardf s溶液，0.5% SDS，和100微克/毫升變性•鮭魚*** DNA之中於42°C預雜合2_4小時。然後在5X SSPE，50%甲醯胺，2X Denhardt’s溶液，0.1% SDS，100微克/毫升變性鮭魚***DNA，和5毫微克/毫升經標記探測核酸中於42°C下進行吸潰雜合18-24小時。之後將該吸潰體在2X SSC中於室溫下洗10分鐘，在IX SSC中於50°C下洗10分鐘，然後在0.5X -41 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（39 ) SSC中洗10-15分鐘。使用衍生自老鼠基因的探測核酸，於幾種組織，包括腎，肝，胎盤和心，之中偵檢到具有約2.4 kb的相對分子量之主要mRNA物種。在腎中偵檢到最高水平。在骨骼肌和脾中偵檢到約4· 5和7.5 kb的大mRNA物種。於人胎組織中，腎經發現可表現相當高水平的2.4 kb mRNA。使用人類探測核酸時 (參看下文）。於相同的這些組織中只偵檢到2.4 kb轉錄本。此外也在淋巴節，胸腺，脾和闌尾中偵檢到相當高水平的 2.4 kb轉錄體。用老鼠和人類兩種促骨堆積素基因偵檢到的轉錄體尺寸都幾乎與老鼠pBl.l FRI-1***體的長度相同，因此可推測其爲一全長度cDNA株。實施例3 OPG在導入外來基因的小鼠體内之系統性輸送使用老鼠OPG株pBl.l作爲PCR的模版以擴增密碼區再分殖到ApoE·肝特定性表現媒體之中（Simonet et al. J. Clin. Invest. £1，1310-1319(1994)，和 PCT 申請第 US94/11675 和共同擁有的美國申請序號08/221,767)。下面的5’和3,寡核甞酸引子分別用於PCR擴增之中： 5丨-GACTAGTCCCACAATGAACAAGTGGCTGTG-3· (SEQIDNO ： 9) 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) S'.ATAAGAATGCGGCCGCTAAACTATGAAACAGCCCAGTGACCATTC-S' (SEQIDNO ： 10) 將PCR反應混合物（Boehringer-Mannheim)依下述處理之： 94°C 1 分鐘，1循環；94°C 20秒，62Ό 30秒，和 74°C 1 分鐘，2 5個循環。擴增之後，以Qiagen PCR管柱純化樣品並 -42- I紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐) 1221482 A7 B7 五、發明説明（4〇 ) 用Spe I和Not I限制酶消化整個晚上。將消化過的產物_ 取，沈澱再分殖到ApoE啓動基因表現媒體中。於微逢射苹得株，HE-OPG，之前，先將其定序以確定其不含突變。將HE-OPG質體係經兩次CsCl密度梯度離心予以純化純化過的質體DNA用Xhol和Ase I消化並將3·6处導外來經濟部中央標準局員工消費合作社印製基因***體以凝膠電泳予以純化。將純化過的片段稀釋在^ mM Tris，pH 7·4，〇·2 mM EDTA中成爲1微克/毫升的悔‘ 5 、、审存注射液。基本上依（Brinster et al·，Proc. Natl. Acad· f l· IJSa §2, 433 8 (1985))。中所述注射取自BDF1 XBDF1-育穑， A 單細胞胚胎，不過，注射針在使用前係經切成斜角Ώ ν 酮處理過。將胚胎置於C02保溫箱中培養過夜後，歡t 听1 5至 20個2-細胞胚胎轉移到假孕的CD1雌小鼠之輸卵管内。在足孕之後，從微注射胚胎移植得到4 9子小鼠。對該子鼠以PCR擴增整合到基因DNA樣品中的外來基因進行筛選。擴增反應的標的區爲包含在表現媒體中的人類Apo E介人子 (intron)所含3 69 pb區。該PCR擴增所用的寡核苷酸爲： 5'- GCC TCT AGA AAG AGC TGG GAC-3* (SEQIDNO ： 11) 5·· CGC CGT GTT CCATTT ATGAGC-3· (SEQIDNO ： 12) PCR的條件爲：94X： 2分鐘，一循環；94°C 1分鐘，63°C 20秒，和72°C 30秒，3 0個循環。於49個原子代中，有9個經鑑定爲PCR陽性導入外來基因建立者（founders)。在8-10週年齡時，取5隻導入外來基因建立者（2, 11， 16，17和28)和5隻對照者（1，12，15，18和30)予以犧牲供驗屍和病理學分析所用。從剩餘的4隻建立者以部份肝切除術 -43- 本紙張尺度適用中國國家標準（CNS ) A4規格（21 OX 297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂. 1221482 A7 B7 五、發明説明（41 ) 分離出肝。在進行部份肝切除術時，將小鼠麻醉並用手術取出一葉肝。從所述全部外來基因建立者；和5雙陰性對照組同窩出生者的肝分離出總細胞RNA(McDonald et al. Meth. Enzymol. 152, 219 (1987))。對這些樣品進行北方氏吸潰分析以評估外來基因表現水平。約1 0微克來自各動物肝的總 RNA經電泳變性凝膠（Ogden et al· Meth. Enzymol 152., 61 (1987))離析後，轉移到HYBOND-N耐綸膜（Amersham)並以 32P dCTP-標記pBl.l***體DNA作爲探測核酸。在50%甲醯胺，5 X SSPE，0.5% SDS，5 X Denhardt’s溶液，100微克 / 毫升變性鮭魚***NDA和2-4 X 106 cmp的標誌探測核酸/毫升雜合緩衝液中於42°C下進行雜合整個晚上。於雜合後，將吸清點置於2XSSC，0.1% SDS中於室溫下各洗5分鐘兩次，接著在0.1 XSSC，0.1% SDS中於55°C下各洗5_10分鐘兩次。建立者和對照組同窩出生小鼠體内導入基因的表現係在放射自顯術後測定的。經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 北方氏吸潰數據顯示出導入外來基因的建立者之中有七隻表現出可偵檢水平的導入基因mRNA(動物編號2，11，16， 17，22，33，和45)。陰性對照小鼠和一隻建立者（#28)未表現出與導入基因相關的mRNA。由於OPG係經預期爲分泌蛋白質，因此導入基因mRNA的過度表現應該可作爲系統性輸送基因產物水平的代表。於PCR和北方氏吸潰陽性小鼠中，動物2，17和22表現出最高水平的外來基因mRNA，且可能顯示出對宿主細胞和組織的更廣遠生物學影響。實施例4 -44- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（42 ) OPG的生物活性將5隻導入外來基因小鼠（動物#2，u，ΐό，17和28)和$隻對月、、組同窩出生小鼠（動物#1，12，15，18和3〇)犧牲以供使用下述程序進行驗屍和病理學分析。在安逸死之前，先將所有動物的鑑別號碼核對過，再予稱重，麻醉和抽血。血腹係以血清和全血液形式保存起來供完全血清化學和血液學=查所用。剛好在用致死c〇2予以最終麻醉之後及大解剖之前進行放射攝影。其後，取出組織並固定在1〇%緩衝一福馬林中供組織學檢驗所用。所收集的組織包括肝，脾，胰，胃，十二指腸，迴腸，結腸，腎，生殖器官，皮膚和乳腺，骨，腦，心，肺，胸腺，氣管，食道，甲狀腺，空腸，盲腸，直腸，腎上腺，膀胱，和骨骼肌。在固定之則，測定肝，胃，腎，腎上腺，髀，和胸腺的全器官重量。於固定之後，將組織處理成石蠟塊，且取得3微米切片。用甲酸溶液將骨組織脱鈣並用蘇木素和曙紅染色所有切片。此外’對某些組織也用Gomoris氏網硬蛋白和Masson，s 氏trichrome進行染色。另外實施酵素組織化學來測定酒石酸鹽抗拒性酸性磷酸酶（TRAP)的表現，該酵素係被蝕骨細胞所高度表現者’該細胞爲單核細胞-巨嗤細胞系的多核骨吸除性細胞。此外也進行BrdU和F480單核細胞-巨噬細胞表面抗原的免疫組織化學來分別偵檢複製細胞和單核細胞-巨噬細胞系。要偵檢F480表現抗原表現時，先將經福馬林固定，石蠟包埋的4微米切片予以脱石蠟並用去離子水予以水合。將該切片用3%過氧化氫驟凝，以Protein Block -45- 本紙張尺度適用中國國家標準（CNS ) A4規格（210x297公釐） (請先閲讀背面之注意事項再填寫本頁) 訂痛· 1221482 A7 B7 五、發明説明（43 ) 經濟部中央標準局員工消費合作社印製 ((Lipshaw，Pittsburgh，PA)予以封阻，並在老鼠單株抗小鼠 FMO (Harlan，Indianapolis，IN)中培育。將此抗體以經生物素化兔子抗-老鼠免疫球蛋白，過氧化酶拼合鏈抗生物素蛋白（BioGenex San Ramon，CA)在DAB作爲 Chr0magen (BioTek，Santa Barbara, CA)之下予以债檢。在内臟組織的大解剖和觀察之下，在導入外來基因的表現子或對照組同窩小鼠中都未發現有異常。器官重量分析顯 π導入外來基因的小鼠之脾尺寸相對於對照組增加約38%。於導入外來基因的表現子體内有稍微擴大的血小板尺寸和增加的循環未染色細胞。在導入外來基因的表現子體内之血小板水平有邊際減少。此外，在導入外來基因表現子中，其血清尿酸，脲蛋，和鹼性磷酸酶水平都傾向於較爲低。琢等表現子經發現具有增高的骨骼放射密度，包括長股（股骨），椎骨，和扁平骨（骨盆骨）。表現子内的股骨之相對尺寸與對照小鼠沒有不同。來自OPG表現子的骨染色切片所得組織學分析顯示出嚴重的骨質石化病，在股骨骨幹中的小梁骨内可看到含有來自初η鬆質的軟骨殘留物。於股骨切片中沒有清晰明確的皮質可鑑別出來。於正常動物中，中央骨幹都填充著骨髓。椎丹切片也顯示出骨質石化性變化，暗示著〇pG_謗導的骨骼變化係系統性者。殘留骨髓顯示出主要爲骨髓要素。其中含有巨核細胞。網硬蛋白染色未顯示出網硬蛋白沈積跡象。對F480-由小鼠體内單核細胞-巨噬細胞衍化的細胞所表現出的一種細胞表面抗原·進行免疫組織化學顯示出在骨髓OPG mRNA expression pattern in tissues The 32P-dCTP-labeled FRI-1 PCR product was used as the detection nucleic acid for duplex human tissue Clonetech to detect the size of human transcripts and determine the expression pattern. Beibu's aspiration was pre-hybridized in 5X SSPE, 50% formamidine, 5X Denhardf's solution, 0.5% SDS, and 100 μg / ml denatured salmon salmon sperm DNA at 42 ° C for 2 to 4 hours. Then hybridize in 5X SSPE, 50% formamidine, 2X Denhardt's solution, 0.1% SDS, 100 μg / ml denatured salmon sperm DNA, and 5 ng / ml labeled probe nucleic acid at 42 ° C. 18 -24 hours. After that, wash the soaked body in 2X SSC at room temperature for 10 minutes, in IX SSC at 50 ° C for 10 minutes, and then in 0.5X -41-this paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 mm) 1221482 A7 B7 V. Description of the invention (39) Wash in SSC for 10-15 minutes. Using probe nucleic acids derived from mouse genes, major mRNA species with relative molecular weights of approximately 2.4 kb were detected in several tissues, including kidney, liver, placenta, and heart. The highest level was detected in the kidney. Large mRNA species of approximately 4.5 and 7.5 kb were detected in skeletal muscle and spleen. In human fetal tissue, the renal meridian was found to exhibit a rather high level of 2.4 kb mRNA. When using human probing nucleic acids (see below). Only 2.4 kb transcripts were detected in the same tissues. In addition, quite high levels of 2.4 kb transcripts were detected in the lymph nodes, thymus, spleen, and appendix. The transcripts detected by both mouse and human osteotropin genes were almost the same length as the mouse pBl.l FRI-1 insert, so it can be speculated to be a full-length cDNA strain. Example 3 Systemic Delivery of OPG in Mice Introduced Foreign Genes The mouse OPG strain pBl.l was used as a template for PCR to amplify the coding region and then cloned into ApoE · liver specific expression media (Simonet et al J. Clin. Invest. £ 1,1310-1319 (1994), and PCT Application No. US94 / 11675 and Co-owned U.S. Application Serial No. 08 / 221,767). The following 5 'and 3, oligonucleotide primers are used for PCR amplification: 5 丨 -GACTAGTCCCACAATGAACAAGTGGCTGTG-3 · (SEQIDNO: 9) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the note on the back first) Please fill in this page for more details) S'.ATAAGAATGCGGCCGCTAAACTATGAAACAGCCCAGTGACCATTC-S '(SEQIDNO: 10) Process the PCR reaction mixture (Boehringer-Mannheim) as follows: 94 ° C for 1 minute, 1 cycle; 94 ° C for 20 seconds, 62Ό 30 Seconds, and 1 minute at 74 ° C, 25 cycles. After amplification, the samples were purified using Qiagen PCR columns and the paper size of -42-I was in accordance with Chinese National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 V. Description of the invention (4〇) Restricted by Spe I and Not I Enzymatic digestion all night. The digested product was taken and the pellet was recolonized into the ApoE promoter gene expression media. Yu Weifeng Sheping obtained the strain, HE-OPG, before sequencing it to make sure it does not contain mutations. The HE-OPG quality system was purified by two CsCl density gradient centrifugations. The purified plastid DNA was digested with Xhol and Ase I and 3, 6 spots were imported to the Central Consumers Bureau of the Ministry of Foreign Affairs to print gene inserts for cloning. Purified by gel electrophoresis. The purified fragments were diluted in ^ mM Tris, pH 7.4, 0.2 mM EDTA to become 1 μg / ml, and the injection solution was stored. Basically (Brinster et al., Proc. Natl. Acad. Fl. IJSa § 2, 433 8 (1985)). The injection described in the above was taken from BDF1 XBDF1-Yu Zhi, A single cell embryo, however, the injection needle was treated with beveled Ώ ν ketone before use. After the embryos were cultured overnight in a C02 incubator, 15 to 20 2-cell embryos were transferred to the fallopian tubes of CD1 female mice that were pseudopregnant. After foot pregnancy, 49 daughter mice were obtained from microinjected embryo transfer. This mouse was screened for foreign genes integrated into the gene DNA sample by PCR amplification. The target region of the amplification reaction is the 3 69 pb region contained in the human Apo E intron included in the expression media. The oligonucleotide used for the PCR amplification is: 5'- GCC TCT AGA AAG AGC TGG GAC-3 * (SEQIDNO: 11) 5 ·· CGC CGT GTT CCATTT ATGAGC-3 · (SEQIDNO: 12) The conditions of the PCR are : 94X: 2 minutes, one cycle; 94 ° C for 1 minute, 63 ° C for 20 seconds, and 72 ° C for 30 seconds, 30 cycles. Of the 49 atomic generations, 9 were found to be PCR-positive into foreign gene founders. At the age of 8-10 weeks, 5 introduced foreign gene founders (2, 11, 16, 17, and 28) and 5 controls (1, 12, 15, 18, and 30) were sacrificed for autopsy and pathology Used for analysis. Partial hepatectomy from the remaining 4 founders-43- This paper size applies the Chinese National Standard (CNS) A4 specification (21 OX 297 mm) (Please read the precautions on the back before filling this page). 1221482 A7 B7 5. Description of the invention (41) Liver is isolated. During partial hepatectomy, mice were anesthetized and one leaf of the liver was surgically removed. Total cellular RNA was isolated from the livers of all the founders of foreign genes; and the littermates of 5 pairs of negative control groups (McDonald et al. Meth. Enzymol. 152, 219 (1987)). Northern samples were analyzed for these samples to assess the level of foreign gene expression. Approximately 10 micrograms of total RNA from liver of each animal was isolated by electrophoretic denaturing gel (Ogden et al. Meth. Enzymol 152., 61 (1987)), and then transferred to HYBOND-N nylon membrane (Amersham) and treated with 32P dCTP. -Label pBl.l insert DNA as probe nucleic acid. In 50% formamidine, 5 X SSPE, 0.5% SDS, 5 X Denhardt's solution, 100 μg / ml denatured salmon sperm NDA and 2-4 X 106 cmp marker detection nucleic acid / ml hybrid buffer at 42 ° C Under heterozygosity all night. After hybridization, the blotting point was placed in 2XSSC, 0.1% SDS and washed twice each at room temperature for 5 minutes, and then washed twice in 0.1 XSSC, 0.1% SDS and 55 ° C twice each at 5 ° C. The expression of the genes introduced into the littermates of the founders and controls was measured after autoradiography. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). The North's sucking data shows that seven of the founders who introduced foreign genes showed detectable levels of imported genes. mRNA (animal numbers 2, 11, 16, 17, 22, 33, and 45). Negative control mice and one founder (# 28) did not show mRNA related to the introduced gene. Since OPG is expected to secrete protein, the overexpression of the introduced gene mRNA should be representative of the level of systemic delivery of gene products. Among PCR- and Northern blot-positive mice, animals 2, 17, and 22 exhibit the highest levels of foreign gene mRNA and may show a broader biological effect on host cells and tissues. Example 4 -44- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297mm) 1221482 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (42) The biological activity of OPG will be 5 Only foreign mouse (animal # 2, u, ΐό, 17 and 28) were introduced and only mice born to the moon, and the same litter (animal # 1, 12, 15, 18, and 30) were sacrificed for use The following procedure was performed for autopsy and pathological analysis. Prior to death, all animal identification numbers were checked, weighed, anesthetized, and blood drawn. Blood The abdominal system is stored in serum and whole blood for complete serum chemistry and hematology = investigation. Radiography was performed just after final anesthesia with lethal CO2 and before macrodissection. Thereafter, the tissue was removed and fixed in 10% buffered formalin for histological examination. The collected tissues include liver, spleen, pancreas, stomach, duodenum, ileum, colon, kidney, reproductive organs, skin and breast, bone, brain, heart, lung, thymus, trachea, esophagus, thyroid, jejunum, cecum, rectum, Adrenal, bladder, and skeletal muscle. During fixation, total organ weights of the liver, stomach, kidney, adrenal glands, iliac crest, and thymus were measured. After fixation, the tissue was processed into paraffin blocks and 3 micron sections were obtained. Bone tissue was decalcified with formic acid solution and all sections were stained with hematoxylin and eosin. In addition, certain tissues were also stained with Gomoris reticulin and Masson's trichrome. In addition, enzyme histochemistry was performed to determine the performance of tartrate-resistant acid phosphatase (TRAP). This enzyme is highly expressed by osteoclasts. This cell is a multinucleated bone-removing cell of monocyte-macrophage cell line . In addition, immunohistochemistry of BrdU and F480 monocyte-macrophage surface antigens was performed to detect replicative cells and monocyte-macrophage cell lines, respectively. To detect the antigenic manifestation of F480, 4 micron sections fixed in formalin and embedded in paraffin were deparaffinized and hydrated with deionized water. This section was quickly coagulated with 3% hydrogen peroxide to Protein Block -45-. This paper size applies Chinese National Standard (CNS) A4 (210x297 mm) (Please read the precautions on the back before filling this page). · 1221482 A7 B7 V. Description of the invention (43) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ((Lipshaw, Pittsburgh, PA) and blocked in a mouse monoclonal anti-mouse FMO (Harlan, Indianapolis, IN) Incubate. This antibody was debtorized with biotinylated rabbit anti-mouse immunoglobulin, peroxidase-linked chain avidin (BioGenex San Ramon, CA) under DAB as Chromoman (BioTek, Santa Barbara, CA). Under the macro dissection and observation of visceral tissue, no abnormalities were found in the foreign gene-expressing expressor or control group littermates. The organ weight analysis showed that the size of the spleen of the foreign gene-introduced mice was relatively small. Increased by about 38% in the control group. There was a slightly enlarged platelet size and increased circulating unstained cells in the introduced progenitors of foreign genes. Platelet water in the expressed progenitors of introduced foreign genes There is a marginal reduction. In addition, in the introduction of foreign gene expressors, serum uric acid, urea eggs, and alkaline phosphatase levels tend to be lower. Zhuo et al. Have been found to have increased bone radiation density, including long Femur (femur), vertebra, and flat bone (pelvic bone). The relative size of femurs in the expressors is not different from that of control mice. Histological analysis of bone stained sections from OPG expressors showed severe osteopetrosis, The trabecular bone in the femoral shaft can be seen to contain cartilage residues from the primary η cancellous. No clear cortex can be identified in the femoral section. In normal animals, the central backbone is filled with bone marrow. The sections also show petrochemical changes in bone, suggesting systemic changes in the skeletal changes induced by OpG. Residual bone marrow shows mainly bone marrow elements. It contains megakaryocytes. Reticulin staining showed no evidence of reticulin deposition .Immunohistochemistry of F480-a cell surface antigen expressed by monocyte-macrophage-derived cells in mice Shows in the bone marrow

210X297公釐） -46 (請先閱讀背面之注意事項再填寫本頁) 、17 續· .! - - II ϋ— 1221482 A7 五、發明説明（44 ) 空間内含有F480陽性細胞。焦點地，扁平化的剛陽性細胞可以看到係直接鄰接於小梁骨表面上。襯著小梁骨的間葉細胞係扁平化且顯得不具活性者。根據 H&E和TRAP染色’在〇pg表現子的小梁骨表面上罕於發現蝕骨細胞。相反地，在生長板吸除軟骨區中可看到蝕骨細胞及/或破軟骨細胞，但其數目相對於對照組係較低者。此外，在通常具有強烈模塑活性的幹骺端之皮質表面上含有蝕骨細胞。表現子與對照組之間的主要差異在於在椎骨和股骨兩者中的小梁骨蚀骨細胞都有明顯的減少。骨蓄積程度與總肝RNA北方氏吸潰所偵檢到的〇PG導入外來基因的 mRNA水平有直接的相互關聯。 OPG表現子的脾具有增加量的紅髓，其擴張係因增加的造血所致。所有造血系都作用出。在對照組與〇PG表現子兩者中的紅髓内都含有F480陽性細胞。兩個表現子（2號和17號）在肝中具有髓外造血竈且其可能是由骨質硬化性骨髓所致0 在胸腺，淋巴節，胃腸道，胰肝膽道，生殖系統，生殖泌尿系統，皮膚，神經系統，心和主動脈，***，骨骼肌與脂肪都沒有可觀察到的異常情形。經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁} 實施例5 小鼠和人類OPG cDNA的分離利用PCR擴增從小鼠腎cdNA庫（Clontech)分離出對應於小鼠OPG mRNA 5 ·端的CDNA株。寡核苷酸係衍生自老鼠0PG cDNA序列且其係下面所示者： -47- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（45 ) 5,-ATCAAAGGCAGGGCATACTTCCTG-3, 5,-GTTGCACTCCTGTTTCACGGTCTG-3, (SEQIDNO ： 13) (SEQIDNO ： 14) 5,-CAAGACACCTTGAAGGGCCTGATG-3, S'-TAACTTTTACAGAAGAGCATCAGCd， (SEQIDNO ： 15) (SEQIDNO ： 16) 經濟部中央標準局員工消費合作社印製 5,-AGCGCGGCCGCATGAACAAGTGGCTGTGCTGCG-3, (SEQIDNO ： 17) 5'-AGCTCTAGAGAAACAGCCCAGTGACCATTCC-3, (SEQIDNO ： 18) 將此程序所得部份和全長度cDNA產物予以定序。將全長產物手Not I和Xba I消化後直接選殖在質體媒體PRCCMV (Invitrogen)内。所得質體稱爲pRcCMV-Mu-OPG。將選殖產物的核苷酸序列與老鼠OPG cDNA序列比對。於跨越〇pg LORF的1300 bp内，老鼠和小鼠DNA序列有約88%相同率。小鼠cDNA序列含有一段401胺基酸LORF，其在與老鼠〇pg 蛋白質序列比對之下發現有〜94%相同率且無間隙。此點顯示所分離出的小鼠cDNA序列編碼小鼠OPG蛋白質，且其序列和構造在整個進化中都高度地保存著。小鼠OPG蛋白質序列在其N-端含有一相同的推定性信號肽，且所有4個潛在的 N -鍵聯糖基化部位都保存著。使用下列老鼠-特定性寡核苷酸從人類腎cDNA庫選殖出一種部份人類OPG cDNA : 5*-GTGAAGCTGTGC AAGAAC CTGATG-3* (SEQIDNO · 19) 5'-ATC AAA GGC AGG GCA TAC TTC CTG-3* (SEQ ID NO ： 20) 將該PCR產物定序並用來設計引子以用於以入中的人類 -48- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 1221482 經濟部中央標準局員工消費合作社印製 A 7 B7 五、發明説明（46 ) OPG基因組株作爲模版所進行的人類cDNA 31端之擴增中： 5*-TCCGTAAGAAACAGCCCAGTGACC-3· (SEQIDNO ： 29) 5,-CAGATCCTGAAGCTGCTCAGTTTG-3, (SEQIDNO ： 21) 將擴增PCR產物定序，並與5、端序列一起用來設計可用來擴增整個人類OPG cDNA密碼序列之5 ·和3 ’人類一特定性引子： 5,-AGCGCGGCCGCGGGGACCACAATGAACAAGTTG-3, (SEQIDNO ： 22) 5,-AGCTCTAGAATTGTGAGGAAACAGCTCAATGGC-3, (SEQIDNO ： 23) 將該全長度人類PCR產物定序後，用Not I和Xba I直接選殖到質體媒體pRcCMV(Invitrogen)内。所得質體稱爲 pRcCMV-人類· OPG。將選殖產物的核甞酸序列與老鼠和小鼠的OPG cDNA序列比對。於跨越OPG LORF的1300 bp區内，老鼠和小鼠DNA序列與人類OPG cDNA約有78-88%的相同率。該人類OPG cDNA序列也含有一 401胺基酸LORF，將其與老鼠與小鼠蛋白質序列比對。預測的人類OPG蛋白質相對於老鼠和小鼠蛋白質分別有約85%相同率，和〜90%相同率。老鼠，小鼠和人類蛋白質的序列排列比對顯示出彼等在進化中有高度的保守。人類蛋白質經預測具有一 N-端信號肽，和5個潛在的N-鍵聯糖基化部位，其中4個係在老鼠和小鼠OPG蛋白質之間保存住者。小鼠0PG的DNA與預測的胺基酸序列示於圖9A和9B中 ((SEQ ID NO : 122)。人類OPG的DNA與經預測的胺基酸序列示於圖9C和9D中（SEQ ID NO : 124)。老鼠，小鼠和人類OPG胺基酸序列的比較示於圖9E和9F中。 -49- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁)210X297 mm) -46 (Please read the precautions on the back before filling this page), 17 Continued ·!--II ϋ— 1221482 A7 V. Description of the invention (44) The space contains F480 positive cells. In focus, the flattened positive cells can be seen directly adjacent to the trabecular bone surface. Mesenchymal cell lines lined with trabecular bone are flat and appear inactive. According to H & E and TRAP staining, osteoclasts were rarely found on the trabecular bone surface of the 0pg expressor. In contrast, osteoclasts and / or chondrocytes can be seen in the cartilage removal area of the growth plate, but the number is lower than that of the control group. In addition, the cortical surface of the metaphysis, which usually has a strong molding activity, contains osteoclasts. The main difference between the expressor and the control group is that there is a significant reduction in trabecular bone erosion bone cells in both the vertebrae and the femur. The degree of bone accumulation is directly related to the mRNA level of foreign genes introduced by OPG detected by Northern Hepatic Aspiration. The OPG expressor's spleen has an increased amount of red pulp, and its expansion is due to increased hematopoietic. All hematopoietic lines work. F480-positive cells were contained in the red pulp in both the control group and the OPG expressor. Two expressors (No. 2 and No. 17) have extramedullary hematopoietic foci in the liver and may be caused by osteosclerotic bone marrow 0 in the thymus, lymph nodes, gastrointestinal tract, pancreas, hepatobiliary tract, reproductive system, genitourinary system , Skin, nervous system, heart and aorta, breast, skeletal muscle, and fat were not observed. Ministry of Economic Affairs Bureau of Standards staff consumer cooperative printed (Read back surface of the page and then fill} Note Example 5 mice and isolated human OPG cDNA was amplified by PCR embodiment separated from the kidney cdNA library (Clontech) corresponds to the mouse end of OPG mRNA 5 · mouse strains CDNA oligonucleotide sequences based 0PG cDNA derived from the mouse-based and which are shown in the following: this paper -47- applies China national standard scale (CNS) A4 size (210X 297 mm) 1221482 A7 B7 V. Description of the invention (45) 5, -ATCAAAGGCAGGGCATACTTCCTG-3, 5, -GTTGCACTCCTGTTTCACGGTCTG-3, (SEQIDNO: 13) (SEQIDNO: 14) 5, -CAAGACACCTTGAAGGGCCTGATG-3, S'-TAACTTTTACAGAAGAGCATCAGCID, 15 ) (SEQIDNO: 16) Ministry of economic Affairs Bureau of standards employees consumer cooperatives printed 5, -AGCGCGGCCGCATGAACAAGTGGCTGTGCTGCG-3, (SEQIDNO: 17) 5'-AGCTCTAGAGAAACAGCCCAGTGACCATTCC-3, (SEQIDNO: 18) part of this program and the resulting full-length cDNA the product to be sequencing. the full-length product after hand Not I and Xba I digested directly cloned in the plastid media pRCCMV (Invitrogen). the resulting plasmid is referred pRcCMV-Mu-OPG. the production of cloned A nucleotide sequence alignment of the mouse cDNA sequence of OPG. 〇pg LORF within the span of 1300 bp, rats and mice, about the same as the DNA sequences of 88%. Mouse cDNA sequence contains a stretch of 401 amino acids LORF, which It was found to have a ~ 94% identity and no gaps when compared with the mouse 0pg protein sequence. This shows that the isolated mouse cDNA sequence encodes the mouse OPG protein, and its sequence and structure have been highly evolved throughout the evolution. save the mouse OPG protein sequence at its N- terminus contains a putative signal peptide of the same, and all 4 potential N - bond-linked glycosylation sites are preserved following mouse - specific oligonucleotide A partial human OPG cDNA was selected from the human kidney cDNA library: 5 * -GTGAAGCTGTGC AAGAAC CTGATG-3 * (SEQIDNO · 19) 5'-ATC AAA GGC AGG GCA TAC TTC CTG-3 * (SEQ ID NO: 20) the PCR products were sequenced and used to design primers for use in human -48- to the scale of this paper applies China national standard (CNS) A4 size (210X297 mm) (please read the Notes on the back page and then fill in ) 1221482 economic Affairs Bureau of standards employees Co-op Preparation of A 7 B7 V. Description of the invention (46) Amplification of the 31 end of human cDNA by using OPG genomic strain as a template: 5 * -TCCGTAAGAAACAGCCCAGTGACC-3 · (SEQIDNO: 29) 5, -CAGATCCTGAAGCTGCTCAGTTTG-3, (SEQIDNO: 21) the amplified PCR product was sequenced and the 5, end of the sequence designed to be used together to amplify the entire cDNA 5. password human OPG sequences and 3 'human-specific primers a: 5, -AGCGCGGCCGCGGGGACCACAATGAACAAGTTG-3, ( SEQIDNO: 22) 5, -AGCTCTAGAATTGTGAGGAAACAGCTCAATGGC-3, (SEQIDNO: 23) the product after sequencing the full length human PCR, with Not I and Xba I cloned directly into the plasmid media pRcCMV (Invitrogen). The resulting plasmid called pRcCMV- human · OPG. Nuclear Chang acid sequence OPG cDNA sequence cloned mice and mice product of comparison. In the 1300 bp region spanning the OPG LORF, mouse and mouse DNA sequences are approximately 78-88% identical to human OPG cDNA. The human OPG cDNA sequence also contained a 401 amino acid LORF, which was aligned with mouse and mouse protein sequences. The predicted human OPG protein is about 85% identical to mouse and mouse proteins, and ~ 90% identical. The alignment of mouse, mouse, and human proteins shows that they are highly conserved in evolution. Human proteins are predicted to have an N-terminal signal peptide and five potential N-linked glycosylation sites, four of which are preserved between mouse and mouse OPG proteins. The DNA and predicted amino acid sequence of mouse OPG are shown in Figures 9A and 9B ((SEQ ID NO: 122). The DNA and predicted amino acid sequence of human OPG are shown in Figures 9C and 9D (SEQ ID NO: 124). Comparison of mouse, mouse and human OPG amino acid sequences is shown in Figures 9E and 9F. -49- This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read first (Notes on the back then fill out this page)

、1T 1221482 A7 B7 五、發明説明（47 ) 其他人類OPG cDNA株的分離揭露出在圖9 C中所示的 DNA序列之位置1〇3處有一 G至C的改變。此核甞酸改變導致圖9C所示胺基酸序列的位置3處發生天冬胺醯胺取代離胺酸之結果。具有這種改變的各株中所含其餘序列都與圖 9C和9D中所示者相同。實施例6 OPG三次元構造的塑造 OPG的胺基端部份與TNFR超族的所有已知成員所具細胞外部份具有同系性（圖1 C)。在TNFR-相關基因的此區内之最顯著圖式爲一段折疊成突出構造的〜40胺基酸，富含半胱胺酸重複序列（Banner et al· Cell 21，431-445(1993))。此圖式通常顯示成四個（3-6範圍）縱排重複（參看圖1 C)，且係已知涉及配體結合之中者（Beutler and van Huffel Science 264· 667-663(1994))。每一重複體通常含有6個間隔的半胱胺酸殘基，彼等涉及三個域間雙硫鍵之形成，該等雙硫鍵稱爲 SSI，SS2，和SS3(Banner et al·，同上）。於某些受體中，例如TNFR2，CD30和CD40，有某些重複域只含有兩個鏈間雙硫鍵（SS1和SS3)。經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 將人類OPG蛋白質序列經由使用Luethy，et al.，同上所述方法與TNFR1細胞外域外型比對，並用Wisconsin Package， version 8.1(Genetics Computer Group，Madison，WI)的 Pretty Polt程式以圖形顯示出結果（圖10)。該排列比對顯示出明確保存著參與域1-4的形成中之半胱胺酸殘基。然後用此排列，使用已知的p55 TNFR1細胞外域3-D構造（Banner et al·， -50- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公瘦Γ 1221482 A7 B7 五、發明説明（48 ) 同上）作爲模版來構成人類OPG N-端域的三次元（3-D)模型。要做到此點時，要從TNFR1晶體構造座標複製出肽主鏈和相同殘基侧鏈的原子座標。其後，使用LOOK程式 (Molecular Applications Group，Palo Alto，CA)產生***體和不同側鏈的其餘座標。之後用LOOK減低其構形能量到最少而予以精製。以類似其他TNFR族員的方式，也假設OPG係結合到一配體。爲了塑造OPG與其配體的交互作用’使用TNF- 的晶體構造來模擬"OPG配體••的3-D模型。用Molscript(Kraulis， J. Appl· Cryst· M，946-950, 1991)將此數據以圖形顯示（圖 11)。構成具有3 TNF/?和3 OPG分子的OPG/配體複合物模型，其中OPG的相對位置與TNFR1在晶體構造中的位置相同。然後使用下述作法來找出可能與其配體交互作用的OPG 殘基：計算出複合物中所含全部殘基的溶劑可接近部位及一個單一的OPG模型。在複合物中具有不同於在單體中的可親性之殘基可能與配體交互作用。使用此構形將人類和小鼠OPG胺基酸序列再排列比對以突顯出包括每一富含半胱胺酸域1-4的序列（圖12A和12B)。每一域具有可以預測的個別構造特性：經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 域1 含有參與在SS2(C41對C54)和SS3(C44對C62)雙键中的4個半胱胺酸。雖然根據雙硫橋聯明顯沒有SS1鍵，但其在位置 28的守恆酪胺酸相對於TNFR1中的Y20係同系者，其係已知參與與H66的交互作用而有助於域形成者。OPG具有在位置 -51 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（49 ) 75的同系組胺酸，可推測〇PG Y28與H75在原蛋白質中係堆積在一起的，如同系殘基在TNFR1中一般者。所以，這兩殘基可能確實對生物活性具有重要性，且截頭到且超過Y28 的N _端OPG可能具有變更的活性。此外，根據本發明的3次元模型，殘基E34和K43係經預測會與結合配體交互作用的。域2 含有6個半胱胺酸且經預測含有SS1(65對C80)， SS2(C83對C98)和SS3(C87對C105)雙硫鍵。此OPG區也包含一從P66延展到Q91的區，其與可和TNFy5形成密切接觸的TNFR1域2部份相對正（參看上文），且其可與OPG配體交互作用。特另·]者，殘基 P66，H68，Y69，Y70，T71，D72， S73，H75，T76，S77，D78，E79，L81，Y82，P85，V86， K88，E89，L90，及Q91，皆根據我們的構造數據經預測會與結合配體發生作用。域3 含有4個參與SS1(C107對C118)和SS3(C124對C142)雙硫鍵的半胱胺酸，但未參與SS2鍵。根據我們的構造數據，殘基 Ell5，L118和K119皆經預測會與OPG配體交互作用。域4 含有4個參與SS1(C145對C160)和SS3(C166對C185)雙硫键，但類似於域3者，不涉及SS2键之4個半胱胺酸。我們的構造數據預測E153和S155會與OPG配體交互作用。因此，預測的OPG構造模型可鑑別出許多高度守恆且可能對其生物活性具重要性之殘基。 ___ -52- 本紙張尺度適用中國國家標準（CNS )八4規格（21〇>< 297公釐） (請先閲讀背面之注意事項再填寫本頁) 訂 _滅· 1221482 A7 B7 五、發明説明（SO ) 實施例7 在哺乳動物細胞中產生重組分必OPG蛋白質爲測定OPG是否確定爲分泌蛋白質，乃將小鼠〇PG cDNA 作爲標記融合到人類 IgGl F(^(Cap〇n et al Nature 337, 525-531 (1989))，並在人類293纖維母細胞中表現。Fc融合係用媒體PFc-A3進行的。pFc-A3含有編碼人類免疫球蛋白IgG- r 1 重鏈所含Fc部份（Ellison et al.同上）從鉸鏈域或第一胺基酸 (Glu-99)到幾基端的區域且侧接著5，_N〇t I融合部位和3，_Sal工和Xba I部位。經由人類脾cDNA庫（Clontech)的PCR擴增構成質體。PCR反應係以100微升的最後體積進行且含有2單位的 Vent DNA 聚合酶（New England Biolabs)於 20 mM Tris-HCl (pH 8·8)，l〇 mM KC1，10juM(NH4)2SO4，2 mM MgS04， 0.1% Triton X-100，各爲400//M的dNTP和1毫微克要擴增的 cDNA庫與各爲1 "Μ的引子。反應係經由在951 2分鐘起始，接著3 0個循環的95°C 30秒，55°C 30秒和73°C 2分鐘。其5'-引子 5 丨 ATAGCGGCCGCTGAGCCCAAATCTTGTGACAAAACTCAC 3 丨（SEQ IN NO ： 24) 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 中含有緊接IgG-r 1鉸鏈域第一個殘基（G1u-99W端的Not I部位。其3 1引子 5’ TCTAGAGTCGACTTATCATTTACCCGGAGACAGGGAGAGGCTCTT-3· (SEQ IN NO ： 25) 中含有Sal I和Xbal部位。用Not I和Sal I消化該717-bp PCR 產物，用1 %瓊脂糖（FMC Corp·)電泳分離，以Geneclean程 -53- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（51 ) 序（BIO 101，Inc·)純化再選殖到經Not I，Sal I-消化過的 pBluescript II KS 媒體内（Stratagene)。將所得質體内 pFc-A3，的***體，定序而確定PCR反應的傳眞度。用下列兩組引子配對擴增經選殖在質體pRcCMV-MuOPG 内的小鼠cDNA : 配對1 5,-CCTCTGAGCTCAAGCTTCCGAGGACCACAATGAACAAG-3, (SEQ ID NO ： 26) 5,-CCTCTGCGGCCGCTAAGCAGCTTATTTTCACGGATTGAACCTG-3, (SEQ ID NO ： 27) 配對2 5,-CCTCTGAGCTCAAGCTTCCGAGGACCACAATGAACAAG-3, (SEQ ID NO : 28) 5，-CCTCTGCGGCCGCTGTTGCATTTCCTTTCTG-3，(SEQIDNO : 30) 第一配對擴增整個OPG LORF，並造成一 Not I限制部位，其與Fc融合媒體pFcA3中的架構内Not I部位相容。pFcA3係經由將Not I限制部份處理到人類IgGl Fc cDNA所含天冬胺酸殘基216的5,端而製備成的。此構造導入一聯結子其編碼兩個跨越OPG蛋白質與IgG Fc區之間的接合之無關聯胺基酸。此產物在聯結到F c部份時，即編碼在人類IgGl Fc區所有227胺基酸殘基後面的全部401個OPG殘基（Fl.Fc)。第二引子配對擴增編碼著OPG所含涵蓋其推定配體結合域的前面 180個胺基酸殘基。如上所述者，該V引子造成一人工Not I 限制部位，其可將C-端截頭OPG LORF於蘇胺酸18〇位置處 _ -54- 本紙張尺度適用中國國家標準（Cns ) A4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁) 訂 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（52 ) 直接融合到IgGl Fc或（CT. fc)。聯結OPG殘基401與人類Fc區所含無菌酸殘基221的胺基酸序列接合可經改質如下：編碼人類Fc區所含殘基216-220 的DNA可依下述予以缺失，或可將對應於人類F c區所含 C220的半光胺酸殘基予以突變或絲胺酸或丙胺酸。這些經改質媒體所編碼的OPF-Fc融合蛋白質可轉染到人類293細胞，或CHO細胞内，並依下文所述純化出重組OPG_Fc融合蛋白質。兩種產物皆經導向地選殖到質體媒體pCEP4(Invitrogen) 内。pCEP46含有Epstein-Barr病毒複製源，且能夠在293-EBNA-1細胞内進行附加體複製。母pCEP4，和pCEP4-Fl.Fc 與pGEP4-CT.Fi：諸媒體都經由使用製造商建議的方法月旨染 (lipofected)到293-EBNA-1細胞内。然後將經轉染的細胞於 100微克/毫升潮黴素中進行選擇培養以選擇出供媒體表現用者，並將所得抗藥性培養物生長到會合。然後在無血清培養基内培養該細胞72小時，取出經調理的培養基並以SDS-PAGE分析之。聚丙烯醯胺凝膠的銀染色偵檢出由抗藥性 293培養物所產生的主要經調理培養基蛋白質。於pCEP4-Fl.Fc和pCEP4-CT.Fc經調理培養基中，豐盛地分泌出具預定尺寸的獨特、分離帶（參看圖13B和13C)。該全長度Fc融合蛋白質會蓄積到高濃度，顯示其可能具安定性。兩種Fc融合蛋白質都在西方氏吸潰上以抗-人類IgGl Fc抗體（Pierce)予以偵檢，結果顯示出彼等爲重組0PG產物。將全長度OPG-Fc融合蛋白質以蛋白質-A管柱層析術（Pierce) -55- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 11 ϋ 11111 n I ϋ n ϋ 1^ 11111^ (請先閲讀背面之注意事項再填寫本頁) 1221482 A7 B7 五、發明説明（53 ) (請先閱讀背面之注意事項再填寫本頁) 用製造商建議的程序予以純化。然後基本上依Matsudaira et al·所述（J· Biol· Chem· 2^2, 10-35(1987))對該蛋白質施以自動Edman降解N -端序列分析。於1 9個循環後讀得下列胺基酸序列：1T 1221482 A7 B7 V. Description of the invention (47) Isolation of other human OPG cDNA strains revealed a G to C change at position 103 of the DNA sequence shown in Figure 9C. This nucleotide change resulted in the substitution of aspartame at position 3 of the amino acid sequence shown in Fig. 9C as a result of the substitution of the aspartate. The remaining sequences contained in each strain with this change were the same as those shown in Figs. 9C and 9D. Example 6 Modeling of the three-dimensional structure of OPG The amine-terminal portion of OPG is homologous to the cell exterior of all known members of the TNFR superfamily (Figure 1C). The most prominent pattern in this region of the TNFR-related gene is a ~ 40 amino acid folded into a prominent structure, rich in cysteine repeats (Banner et al. Cell 21, 431-445 (1993)) . This pattern is usually shown as four (range 3-6) tandem repeats (see Figure 1C) and is known to involve ligand binding (Beutler and van Huffel Science 264 · 667-663 (1994)) . Each repeat usually contains 6 spaced cysteine residues, which are involved in the formation of three interdomain disulfide bonds. These disulfide bonds are called SSI, SS2, and SS3 (Banner et al., Supra. ). In some receptors, such as TNFR2, CD30 and CD40, there are certain repeat domains that contain only two interchain disulfide bonds (SS1 and SS3). Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling this page). The human OPG protein sequence was compared with the TNFR1 extracellular domain by using Luethy, et al. The Pretty Polt program of Wisconsin Package, version 8.1 (Genetics Computer Group, Madison, WI) graphically displays the results (Figure 10). This alignment shows that the cysteine residues involved in the formation of domains 1-4 are clearly preserved. Then use this arrangement, using the known p55 TNFR1 extracellular domain 3-D structure (Banner et al., -50- This paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 male thin Γ 1221482 A7 B7 V. Invention Note (48) Ibid.) As a template to construct a three-dimensional (3-D) model of the human OPG N-terminal domain. To do this, the peptide backbone and the same residue side chains are copied from the TNFR1 crystal structure coordinates After that, the LOOK program (Molecular Applications Group, Palo Alto, CA) was used to generate the inserts and the remaining coordinates of the different side chains. The LOOK was then used to reduce its configuration energy to a minimum and was refined. Similar to other TNFR families It also assumes that OPG is bound to a ligand. In order to shape the interaction between OPG and its ligands, the crystal structure of TNF- is used to simulate the "OPG ligand" 3-D model. Molscript (Kraulis, J. Appl. Cryst · M, 946-950, 1991) This data is shown graphically (Figure 11). An OPG / ligand complex model with 3 TNF /? And 3 OPG molecules is constructed, where the relative position of OPG and Position of TNFR1 in crystal structure . Then use the following method to find the OPG residues that may interact with its ligand: calculate the solvent accessible parts of all the residues contained in the complex and a single OPG model. The affinity residues in the monomer may interact with the ligand. Use this configuration to rearrange the human and mouse OPG amino acid sequences to highlight the inclusion of each cysteine-rich domain 1- Sequence of 4 (Figures 12A and 12B). Each field has predictable individual structural characteristics: Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) Field 1 contains participation in SS2 (C41 vs. C54) and SS3 (C44 vs. C62) double cysteine in the double bond. Although there is obviously no SS1 bond according to the disulfide bridge, its conserved tyrosine at position 28 is relative to Y20 in TNFR1 Is a fellow, who is known to participate in the interaction with H66 and contribute to the formation of the domain. OPG has the position -51-This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 Central Ministry of Economic Affairs Printed by Standards Bureau Consumer Cooperatives A7 B7 V. Invention description (49) 75 The homologous histidine acid can be inferred that OPG Y28 and H75 are stacked together in the original protein, as if the residues are ordinary in TNFR1. Therefore, these two residues may be true It is important for biological activity, and the N_terminal OPG truncated to and beyond Y28 may have altered activity. In addition, according to the three-dimensional model of the present invention, residues E34 and K43 are predicted to interact with the binding ligand. Domain 2 contains 6 cysteine and is predicted to contain SS1 (65 vs. C80), SS2 (C83 vs. C98), and SS3 (C87 vs. C105) disulfide bonds. This OPG region also contains a region extending from P66 to Q91, which is relatively positive with the TNFR1 domain 2 portion that can be in close contact with TNFy5 (see above), and it can interact with OPG ligands. Special]], residues P66, H68, Y69, Y70, T71, D72, S73, H75, T76, S77, D78, E79, L81, Y82, P85, V86, K88, E89, L90, and Q91, all Based on our structural data, it is predicted to interact with the binding ligand. Domain 3 contains four cysteines that participate in the disulfide bonds of SS1 (C107 vs. C118) and SS3 (C124 vs. C142), but do not participate in the SS2 bond. Based on our construction data, residues Ell5, L118, and K119 are predicted to interact with OPG ligands. Domain 4 contains four disulfide bonds involved in SS1 (C145 vs. C160) and SS3 (C166 vs. C185), but similar to domain 3, it does not involve the four cysteine acids of the SS2 bond. Our construction data predict that E153 and S155 will interact with OPG ligands. Therefore, the predicted OPG construction model can identify many highly conserved residues that may be important for their biological activity. ___ -52- This paper size is in accordance with Chinese National Standard (CNS) 8-4 specifications (21〇 > < 297mm) (Please read the precautions on the back before filling this page) Order_Off · 1221482 A7 B7 V. Description of the invention (SO) Example 7 Production of heavy component OPG protein in mammalian cells To determine whether OPG is determined to be a secreted protein, mouse PGG cDNA was fused to human IgG1 F (^ (CapOn et al. Nature 337, 525-531 (1989)), and expressed in human 293 fibroblasts. Fc fusions were performed using the media PFc-A3. PFc-A3 contains Fc that encodes human immunoglobulin IgG-r 1 heavy chain The part (Ellison et al. Ibid.) From the hinge domain or the first amino acid (Glu-99) to several bases and flanked by the 5, _Not I fusion site and the 3, _Sal site and Xba I site. PCR amplification of the human spleen cDNA library (Clontech) constituted the plastids. The PCR reaction was performed in a final volume of 100 μl and contained 2 units of Vent DNA polymerase (New England Biolabs) in 20 mM Tris-HCl (pH 8 ·· 8), 10 mM KC1, 10juM (NH4) 2SO4, 2 mM MgS04, 0.1% Triton X-100, each 400 / M dNTP 1 ng of cDNA library to be amplified with primers of 1 " M each. The reaction was initiated at 95 2C for 30 minutes, followed by 30 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds and 73 ° C. 2 minutes. Its 5'-primer 5 丨 ATAGCGGCCGCTGAGCCCAAATCTTGTGACAAAACTCAC 3 丨 (SEQ IN NO: 24) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). It contains IgG-r. The first residue of the hinge domain (Not I site at the end of G1u-99W. Its 3 1 primer 5 'TCTAGAGTCGACTTATCATTTACCCGGAGACAGGGAGAGGCTCTT-3 · (SEQ IN NO: 25) contains the Sal I and Xbal sites. This digestion with Not I and Sal I 717-bp PCR products, separated by 1% agarose (FMC Corp ·) electrophoresis, and Geneclean process-53- This paper size applies Chinese National Standard (CNS) A4 (210X 297 mm) 1221482 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Printed by Consumer Cooperative A7 B7 V. Description of the invention (51) Sequence (BIO 101, Inc.) Purified and then cloned into NotI, Sal I-digested pBluescript II KS media (Stratagene). The obtained pFc-A3, inserts were sequenced to determine the transmission of the PCR reaction. The following two sets of primer pairs were used to amplify the mouse cDNA selected in plastid pRcCMV-MuOPG: Pair 1 5, -CCTCTGAGCTCAAGCTTCCGAGGACCACAATGAACAAG-3, (SEQ ID NO: 26) 5, -CCTCTGCGGCCGCTAAGCAGCTTATTTTCACGGATTGAACCTG-3, (SEQ ID NO : 27) Pair 2 5, -CCTCTGAGCTCAAGCTTCCGAGGACCACAATGAACAAG-3, (SEQ ID NO: 28) 5, -CCTCTGCGGCCGCTCTTGTGCATTTCCTTTCTG-3, (SEQ IDNO: 30) The first pair amplifies the entire OPG LORF and creates a Not I restriction site, which is related to Not I site compatible in the Fc fusion media pFcA3. pFcA3 was prepared by processing the Not I restriction portion to the 5 'end of aspartic acid residue 216 contained in the human IgG1 Fc cDNA. This construct introduces a linker that encodes two unrelated amino acids that span the junction between the OPG protein and the IgG Fc region. This product, when linked to the Fc portion, encodes all 401 OPG residues (Fl.Fc) behind all 227 amino acid residues in the human IgG1 Fc region. The second primer paired amplification encodes the first 180 amino acid residues contained in OPG covering its putative ligand binding domain. As mentioned above, the V primer creates an artificial Not I restriction site, which can place the C-terminal truncated OPG LORF at the position of threonine -18. -54- This paper size applies the Chinese National Standard (Cns) A4 specification (210X 297 mm) (Please read the notes on the back before filling out this page) Order 1221482 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (52) Direct fusion into IgGl Fc or (CT. Fc ). The amino acid sequence joining the OPG residue 401 to the sterile acid residue 221 contained in the human Fc region can be modified as follows: DNA encoding residues 216-220 contained in the human Fc region can be deleted as follows, or The semi-photogenic amino acid residue corresponding to C220 contained in the human F c region is mutated or serine or alanine. The OPF-Fc fusion protein encoded by these modified media can be transfected into human 293 cells or CHO cells, and the recombinant OPG_Fc fusion protein can be purified as described below. Both products were directedly cloned into the plastid media pCEP4 (Invitrogen). pCEP46 contains Epstein-Barr virus replication source and is capable of episomal replication in 293-EBNA-1 cells. Maternal pCEP4, and pCEP4-Fl.Fc and pGEP4-CT.Fi: All media were Lipofected into 293-EBNA-1 cells using methods recommended by the manufacturer. The transfected cells were then selectively cultured in 100 μg / ml hygromycin to select media-expressing users, and the resulting drug-resistant cultures were grown to confluence. The cells were then cultured in a serum-free medium for 72 hours, and the conditioned medium was removed and analyzed by SDS-PAGE. The silver staining of the polyacrylamide gel detected the main conditioned medium protein produced by the drug-resistant 293 culture. In the conditioned media of pCEP4-Fl.Fc and pCEP4-CT.Fc, a unique, separated band of a predetermined size is abundantly secreted (see Figs. 13B and 13C). This full-length Fc fusion protein accumulates to high concentrations, indicating that it may be stable. Both Fc fusion proteins were detected on Western blots with anti-human IgGl Fc antibodies (Pierce), and the results showed that they were recombinant OPG products. The full-length OPG-Fc fusion protein was subjected to protein-A column chromatography (Pierce) -55- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) 11 ϋ 11111 n I ϋ n ϋ 1 ^ 11111 ^ (Please read the notes on the back before filling this page) 1221482 A7 B7 V. Description of the invention (53) (Please read the notes on the back before filling this page) Purify using the procedure suggested by the manufacturer. The protein was then subjected to automatic Edman degradation N-terminal sequence analysis substantially as described by Matsudaira et al. (J. Biol. Chem. 2 ^ 2, 10-35 (1987)). The following amino acid sequence was read after 19 cycles:

NH2-ETLPPKYLHYDPETGHQLL-C02H (SEQ ID NO : 31) 此序列與預測的從胺基酸殘基22開始之小鼠〇pg胺基酸序列相同，可推測自然的哺乳動物前導段切斷部位係在胺基酸殘基Q21-E22，而不是原來所預測的在Y31-D32之間。用pCEP4-Fl.Fc和pCEP4-CT.Fc在293-EBNA細胞中進行的表現實驗發明OPG的一種分泌蛋白質，且可系統地作用以結合其配體。採用類似於構成和表現muOPG[22-180]_Fc和muOPG[22· 400]-Fc融合體所用的程序來產生另外的小鼠和人類OPG-Fc 融合蛋白質。融合到人類IgGl的Fc區之編碼胺基酸1-185的鼠類OPG cDNA[muOPG Ct(185).Fc]係依下述而構成的。用下列引子配對於上述聚合酶連鎖反應中擴增取自質體pRcCMV Mu促骨堆積肽的鼠類OPG cDNA(實施例5中所述者）：經濟部中央標準局員工消費合作社印製 1333-82 ： 5，-TCC CTT GCC CTG ACC ACT CTT-3· (SEQ ID NO ·· 32) 1333-80 : 5，-CCT CTG CGG CCG CAC ACA CGT TGT CAT GTG TTG C-3· (SEQ ID NO : 33) -56- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7五、發明説明（54 ) 此引子配對擴增的是編碼如圖9 A所示OPG讀碼中的胺基酸殘基63_185(對應於bp 278-645)之鼠類OPG cDNA。其3,引子含有一個Not I限制部位，其與F c融合媒體pFcA3的架構内Not I部位相容。其產物也跨越位於bp 436的一獨特ecori 限制部位。將經擴增的PCR產物純化，用Not I和EcoR I切，並純化所得EcoRI_Not I限制片段。將含有鼠類1-401 OPG-Fc 融合***體的媒體pCEP4用EcoRI和Notl切，純化並連接到上面產生的PCR產物。所得以pCEP4爲基的表現媒體編碼緊接在人類IgGl Fc區所含全部227胺基酸殘基之後的OPG殘基 1_185。將鼠類〇pg 1-185，Fc融合媒體轉染到293細胞中，施以藥物選擇培養，並產生調理培養基，如上文所述者。所得分泌鼠類OPG 1-185.Fc融合產物再以蛋白質-A管柱層析術（Pierce)用製造商建議的程序予以純化。依下述構成稠合到人類IgGl所含Fc區且編碼胺基酸殘基卜 194的鼠類OPG DNA( rrmOPG Ct(193).Fc)。用下列引子配對擴增得自質體pRcCMV Mu促骨堆積肽的小鼠OPG cDNA : 1333-82 ： 5，-TCC CTT GCC CTG ACC ACT CTT-3· (SEQ ID NO : 34) 1333-81·· 5f-CCT CTG CGG CCG CCT TTT GCG TGG CTT CTC TGT T-3f (SEQ ID NO ： 35) 此引子配對擴增的是編碼OPG讀碼所含胺基酸殘基70_ 194 (對位於bp 298-672)的鼠類OPG cDNA區。其3 ’引子含有一 Notl限制部位，其可與Fc融合媒體pFcA3的架構内Notl部 (請先閲讀背面之注意事項再填寫本頁) 、?τ _滅· -57- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） 1221482 Μ _Β7__ 五、發明説明（55 ) 位相容。該產物也跨越位於bp 436處的一獨特EcoRI限制部位。經擴增的PCR產物依上文所述經選殖到鼠類〇PG[ 1-401] F c融合媒體内。所得以PCEP4爲基的表現媒體編碼著緊接在人類IgGl Fc區所含全部227胺基酸殘基之後的OPG殘基1· 194。將該鼠類〇PG 1-194.FC融合媒體轉染到293細胞中，予以藥物選擇培養，及產生調理培養基。所得分泌融合產物以蛋白質-A管柱層析術（Pierce)用製造商建議的程序予以純化。編碼著融合到人類IgGl所含Fc區的胺基酸1-401之人類 OPG DNA係依下述構成的。用下列寡核甞酸引子擴增在質體pRcCMV-hu促骨堆積肽中的人類〇PG DNA(實施例5中所述者）： 1254-90 ： 5' CCT CTG AGC TCA AGC TTG GTT TCC GGG GAC CAC ATT G-3· (SEQ ID NO : 36) 1254-95 : 5-fCCT CTG CGG CCG CTA AGC AGC TTA TTT TTA CTG AAT GG_3· (SEQ ID NO : 3 7) 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 所得PCR產物編碼全長度人類OPG蛋白質且造成一Not I限制部位其可與F c融合媒體Fc A3中的架構内Not I部位相容。將該PCR產物依上述導引地選殖到質體媒體PCEP4中。所得表現媒體編碼緊接在人類IgGl Fc區所含227胺基酸殘基之後的人類OPG殘基1-401。從經轉染及藥物選擇培養過的細胞產生調理培養基並以蛋白質-A管柱層析術（Pierce)用製造商建議的程序純化huOPGFl.Fc融合產物。 __ -58- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（56 ) 編碼著融合到人類IgGl Fc區的胺基酸1-201之人類OPG DNA[huOPG Ct(201).Fc]係依下述構成的。用下列寡核甞酸引子配對經由PCR擴增選殖自質體pRrCMV-hu促骨堆積肽的人類 OPGcDNA: 1254-90 ： 5，-CCT CTG AGC TCA AGC TTG GTT TCC GGG GAC CAC AAT G-3f (SEQ ID NO : 3 8 ) 1254-92 : 5，-CCTCTGCGGCCGCCAGGGTAACATCTATTCCAC-3· (SEQ ID NO : 3 9) 此引子配對係擴增編碼OPG讀碼所含胺基酸殘基1-201的人類OPG cDNA區，且在3 ’端造成一 Not I限制部位其可與 F c融合媒FcA3架構内Not I部位相容。此產物於聯結到F c部份時係編碼緊接在人類IgGl Fc區所含全部221胺基酸殘基之後的OPG殘基1·201。依上述將該PCR導引地選殖在質體媒體pCEP4内。從經轉染和藥物選擇培養過的細胞產生經調理培養基，並以蛋白質-A管柱層析術（Pierce)用製造商建議的程序純化huOPGCt(201).Fc融合產物。使用下述程序構成且表現未經融合的小老鼠和人類OPG。經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 供全長度鼠類OPG(殘基1-401)的哺乳動物表現所用之質體係用取自pRcCMV Mu·促骨堆積肽的鼠OPG cDNA***體經RCR擴增產生且選殖到表現媒體pDSR π中（DeClerck et · atl. J. Biol. Chem. 266, 3893 (1991))。使用下列寡核甞酸引子： -59- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（57 ) 1295-26 ： 5f-CCG AAG CTT CCA CCA TGA ACA AGT GGC TGT GCT GC-3，（SEQ ID NO : 40) 1295-27 ： 5，-CCT CTG TCG ACT ATT ATA AGC AGC TTA TTT TCA CGG ATT G-3’ （SEQ ID NO : 4 1 ) 依上述以PCR擴增鼠類OPG全長度讀碼。將PCR產物純化並用内核酸限制酶 Hind III 和 Xba I(Boehringer Mannheim， Indianapolis，IN)在製造商建議的條件下進行消化，然後連接到經Hind III和Xba I消化過的pDSR沈。以内核酸限制酶消化偵檢出重組株後，予以定序以確定在PCR擴增步驟中沒有產生突變。將所得質體，pDSR以-muOPG以鈣媒介轉染導到田鼠卵巢 (CHO)細胞内（Wigler et al. Cell II，233 (1977))。根據質體媒體中的二氫葉酸還原酶（DHFR)基因的表現選擇個別的株系並分離出幾種株系。經由CHO細胞調理培養基的西方氏吸潰分析監測鼠OPG重組蛋白質的表現。選擇出高表現性細胞並依所述（DeClerck et al.，同上）用胺甲碟呤處理以進一步擴增OPG表現。接著從CHO細胞系產生調理培養基以進一步純化重組分泌鼠OPG蛋白質。經由將pRcCMV-hu促骨堆積肽中的cDNA***體直接分殖到媒體pDSR仪中以產生供全長度人類〇PG(胺基酸1-401)哺乳動物表現所用的質體（DeClerck et al·，同上）。將該 pRcCMV-OPG質體用Not I消化到完全，用Klenow予以鈍端 -60- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X 297公釐） (請先閱讀背面之注意事項再填寫本頁) 、τ 1221482 A7 B7 五、發明説明（58 ) 化，再用Xba I消化到完全。將媒體DNA用Hind III消化，用 Klenow鈍端化，再用Xbal消化，之後，連接到〇pg***體。然後將重組質體定序以確定人類OPG cDNA的恰當定向。依上述將所得質體pDSR仪-muOPG導到田鼠卵巢（CHO)細胞内。根據質體媒體所含二氫葉酸還原酶（DHFR)基因的表現選擇出個別株系並分離出幾種株系。經由CHO細胞調理培養基的西方氏吸潰分析監測人類OPG重組蛋白質之表現。選擇出高表現性株系，並用胺甲碟呤處理進一步擴增OPG表現。之後用表現人類OPG的CHO細胞系產生調理培養基供蛋白質純化之用。依下述構成編碼殘基1-185的鼠類OPG之表現媒體。用下列寡核甞酸引子從pRcCMV-Mu OPG擴增鼠類OPG cDNA : 1333-82 : 5丨-TCC CTT GCC CTG ACC ACT CTT-3’（SEQ ID NO : 42) 1356-12 : 5f-CCT CTG TCG ACT TAA CAC ACG TTG TCA TGT GTT GC_3，（SEQ ID NO : 43) 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 續_ 此引子配對係擴增編碼著OPG讀碼中的胺基酸63-185(bp 278-645)之鼠〇PG cDNA區且含有緊接在半胱胺酸密碼子 (C185)後的人造停止密碼，其後接著一人造Sal I核酸内切限制酶部位。預期的產物含有可用來分殖到預存在媒體中的内Eco RI限制部位。於PCR擴增後，用Eco RI和Sal I核酸内切限制酶切所得純化產物，並以凝膠純化大片段。之後將純化產物分殖到上述pBluescript_muOPG Fl.Fc的Eco RI和Sal -61 -NH2-ETLPPKYLHYDPETGHQLL-C02H (SEQ ID NO: 31) This sequence is the same as the predicted pg amino acid sequence of the mouse starting from amino acid residue 22, and it can be speculated that the natural mammalian leader cut site is in the amine Acid residues Q21-E22 instead of Y31-D32 as originally predicted. The expression experiments performed with pCEP4-Fl.Fc and pCEP4-CT.Fc in 293-EBNA cells invented a secreted protein of OPG, and it can act systematically to bind its ligand. A procedure similar to that used to construct and express muOPG [22-180] _Fc and muOPG [22 · 400] -Fc fusions was used to generate additional mouse and human OPG-Fc fusion proteins. The murine OPG cDNA [muOPG Ct (185) .Fc] encoding amino acid 1-185 fused to the Fc region of human IgG1 was constructed as follows. The following primers were used to amplify the murine OPG cDNA (described in Example 5) from the plastid pRcCMV Mu bone accumulation promoting peptide in the above polymerase chain reaction: printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 1333 82: 5, -TCC CTT GCC CTG ACC ACT CTT-3 · (SEQ ID NO ·· 32) 1333-80: 5, -CCT CTG CGG CCG CAC ACA CGT TGT CAT GTG TTG C-3 · (SEQ ID NO: 33) -56- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 Printed by A7 B7, Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (54) Murine OPG cDNA encoding amino acid residues 63-185 (corresponding to bp 278-645) in the OPG reading shown in Figure 9A. Third, the primer contains a Not I restriction site, which is compatible with the Not I site in the framework of the F c fusion medium pFcA3. Its product also spans a unique ecori restriction site at bp 436. The amplified PCR product was purified, cut with Not I and EcoR I, and the resulting EcoRI_Not I restriction fragment was purified. The media pCEP4 containing the murine 1-401 OPG-Fc fusion insert was cut with EcoRI and Notl, purified and ligated to the PCR product generated above. The resulting expression media based on pCEP4 encodes OPG residues 1-185 immediately after all 227 amino acid residues contained in the human IgG1 Fc region. Murine Opg 1-185, Fc fusion media was transfected into 293 cells, drug-selective cultures were applied, and conditioning media were generated, as described above. The resulting secreted murine OPG 1-185.Fc fusion product was purified by protein-A column chromatography (Pierce) using procedures recommended by the manufacturer. A murine OPG DNA (rrmOPG Ct (193) .Fc) fused to the Fc region contained in human IgG1 and encoding amino acid residue 194 was constructed as follows. The mouse OPG cDNA obtained from plastid pRcCMV Mu osteotrophic peptide was amplified using the following primer pairs: 1333-82: 5, -TCC CTT GCC CTG ACC ACT CTT-3 · (SEQ ID NO: 34) 1333-81 · 5f-CCT CTG CGG CCG CCT TTT GCG TGG CTT CTC TGT T-3f (SEQ ID NO: 35) This primer pair amplifies the amino acid residues 70_ 194 (pairs located at bp 298- 672) murine OPG cDNA region. The 3 'primer contains a Notl restriction site, which can be fused to the Fc fusion media pFcA3 Notl within the framework (please read the precautions on the back before filling out this page),? Τ _OFF · -57- This paper is applicable to China Standard (CNS) A4 specification (210 × 297 mm) 1221482 Μ_Β7__ 5. Description of the invention (55) is compatible. This product also spans a unique EcoRI restriction site at bp 436. The amplified PCR products were selected into murine OPG [1-401] F c fusion media as described above. The obtained PCEP4-based expression media encodes OPG residue 1.194 immediately after all 227 amino acid residues contained in the human IgG1 Fc region. The murine OPG 1-194.FC fusion medium was transfected into 293 cells, drug-selectively cultured, and a conditioning medium was produced. The resulting secreted fusion product was purified by protein-A column chromatography (Pierce) using procedures recommended by the manufacturer. The human OPG DNA encoding amino acids 1-401 fused to the Fc region contained in human IgG1 was constructed as follows. Human OPG DNA in plastid pRcCMV-hu bone accumulation peptide (described in Example 5) was amplified using the following oligonucleotide primers: 1254-90: 5 'CCT CTG AGC TCA AGC TTG GTT TCC GGG GAC CAC ATT G-3 · (SEQ ID NO: 36) 1254-95: 5-fCCT CTG CGG CCG CTA AGC AGC TTA TTT TTA CTG AAT GG_3 · (SEQ ID NO: 3 7) Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Printed (please read the notes on the back before filling out this page) The PCR product obtained encodes a full-length human OPG protein and causes a Not I restriction site that is compatible with the Not I site in the framework of F c fusion media Fc A3. The PCR product was cloned into the plastid medium PCEP4 as described above. The resulting expression media encodes human OPG residues 1-401 immediately following the 227 amino acid residue contained in the human IgG1 Fc region. Conditioned medium was generated from transfected and drug-selected cultured cells and the huOPGFl.Fc fusion product was purified by protein-A column chromatography (Pierce) using the procedure recommended by the manufacturer. __ -58- This paper size applies Chinese National Standard (CNS) A4 (210X 297 mm) 1221482 A7 B7 V. Description of the invention (56) Human OPG encoding amino acid 1-201 fused to human IgGl Fc region DNA [huOPG Ct (201) .Fc] has the following structure. Human OPG cDNA cloned from plastid pRrCMV-hu osteoporosis peptide was amplified by PCR using the following oligonucleotide primer pairing: 1254-90: 5, -CCT CTG AGC TCA AGC TTG GTT TCC GGG GAC CAC AAT G-3f (SEQ ID NO: 3 8) 1254-92: 5, -CCTCTGCGGCCGCCAGGGTAACATCTATTCCAC-3 · (SEQ ID NO: 3 9) This primer pairing system amplifies human OPG encoding amino acid residues 1-201 contained in the OPG reading code The cDNA region and a Not I restriction site at the 3 ′ end are compatible with the Not I site in the F c fusion media FcA3 framework. This product, when linked to the Fc portion, encodes OPG residue 1.201 immediately after all 221 amino acid residues contained in the human IgG1 Fc region. This PCR was guided to colony in pCEP4, a plastid vector, as described above. Conditioned medium was generated from transfected and drug-selected cultured cells, and the huOPGCt (201) .Fc fusion product was purified by protein-A column chromatography (Pierce) using procedures recommended by the manufacturer. The following procedure was used to construct and express unfused mouse and human OPG. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling out this page) The quality system for mammalian performance of full-length mouse OPG (residues 1-401) was taken from pRcCMV Mu The osteoplastin murine OPG cDNA insert was generated by RCR amplification and cloned into the expression media pDSR π (DeClerck et. Atl. J. Biol. Chem. 266, 3893 (1991)). The following oligonucleotide primers were used: -59- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 × 297 mm) 1221482 Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 5. Invention Description (57) 1295- 26: 5f-CCG AAG CTT CCA CCA TGA ACA AGT GGC TGT GCT GC-3, (SEQ ID NO: 40) 1295-27: 5, -CCT CTG TCG ACT ATT ATA AGC AGC TTA TTT TCA CGG ATT G-3 ' (SEQ ID NO: 4 1) The murine OPG full-length reading code was amplified by PCR as described above. The PCR product was purified and digested with the endonucleic acid restriction enzymes Hind III and Xba I (Boehringer Mannheim, Indianapolis, IN) under conditions recommended by the manufacturer, and then connected to a pDSR pellet digested with Hind III and Xba I. After detection of the recombinant strain by digestion with the internal nucleic acid restriction enzyme, sequencing was performed to confirm that no mutation was generated during the PCR amplification step. The resulting plastids, pDSR, were transduced with -muOPG and calcium media into vole ovary (CHO) cells (Wigler et al. Cell II, 233 (1977)). Individual strains were selected based on the performance of the dihydrofolate reductase (DHFR) gene in plastid media and several strains were isolated. The expression of murine OPG recombinant protein was monitored by Western blot analysis of CHO cell conditioning medium. Highly expressive cells were selected and treated with methotrexate as described (DeClerck et al., Supra) to further expand OPG performance. Conditioning medium was then generated from the CHO cell line to further purify the recombinant secreted murine OPG protein. The cDNA insert in pRcCMV-hu osteotropin was cloned directly into the media pDSR instrument to generate plastids for full-length human OPG (amino acid 1-401) mammalian expression (DeClerck et al. , Ibid.). The pRcCMV-OPG plastid was completely digested with Not I and blunt-ended with Klenow-60. This paper size is applicable to China National Standard (CNS) A4 (21〇X 297 mm) (Please read the notes on the back first Fill out this page again), τ 1221482 A7 B7 V. Description of the invention (58), and then digest it with Xba I completely. The media DNA was digested with Hind III, blunt-ended with Klenow, and digested with Xbal, after which it was ligated to the 0pg insert. The recombinant plastids were then sequenced to determine the proper orientation of the human OPG cDNA. The resulting plastid pDSR meter-muOPG was introduced into the voles (CHO) cells of the voles as described above. Based on the expression of the dihydrofolate reductase (DHFR) gene contained in the plastid media, individual strains were selected and several strains were isolated. The performance of human OPG recombinant protein was monitored by Western blot analysis of CHO cell conditioning medium. Highly expressive strains were selected and treated with methotrexate to further expand OPG performance. A CHO cell line expressing human OPG was then used to generate a conditioning medium for protein purification. The expression media of murine OPG encoding residues 1-185 are constructed as follows. Murine OPG cDNA was amplified from pRcCMV-Mu OPG using the following oligonucleotide primers: 1333-82: 5 丨 -TCC CTT GCC CTG ACC ACT CTT-3 '(SEQ ID NO: 42) 1356-12: 5f-CCT CTG TCG ACT TAA CAC ACG TTG TCA TGT GTT GC_3, (SEQ ID NO: 43) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) Continued _ This primer pairing system is amplified The mouse PG cDNA region encoding the amino acid 63-185 (bp 278-645) in the OPG reading and contains the artificial stop code immediately after the cysteine codon (C185), followed by an artificial Sal I endonuclease restriction site. The expected product contains internal Eco RI restriction sites that can be used to colonize into pre-existing media. After PCR amplification, the resulting purified product was digested with Eco RI and Sal I endonuclease restriction enzymes, and large fragments were purified by gel. The purified product was then cloned into Eco RI and Sal -61 of pBluescript_muOPG Fl.Fc as described above-

本紙張尺度賴t國國玉系準（CNS ) A4規格（210X297公楚T 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（59 ) I消化物之大限制片段。用Hind III和Xho I消化所得質體並以凝膠純化出小片段。接著將含有編碼1-185開放讀碼的該片段分殖到表現媒體pCEP4的Hind III和Xho I消化體内。所得媒體，pmuOPG[l-185]編碼著在位於185位置的半胱胺酸殘基終止之截頭OPG多肽。依上述從經轉染和經藥物選擇培養過的細胞產生經調理培養基。 1333-82 ·· 5，_TCC CTT GCC CTG ACC ACT CTT _3· (SEQ ID NO ： 44) 1356-13 ： 5'-CCT CTG TCG ACT TAC TTT TGC GTG GCT TCT CTG TT-3，（SEQ ID NO : 45) 此引子配對係擴增編碼OPG讀碼中的胺基酸70-194(bp 298-672)的鼠類OPG cDNA區且含有緊接著離胺酸密碼子 (K194)後會的人造停止密碼，其後接著人造Sal I核酸内切限制部位。預期的產物含有可用來分殖到預先存在的媒體中之内Eco RI限制部位。於PCR擴增後，用Eco RI和Sal I核酸内切限制酶切所得純化產物，並以凝膠純化出大片段。之後將純化產物分殖到上述pBluescript-muOPG Fl.Fc的Eco RI和Sal I消化體之大限制片段。所得質體經Hind III和Xho I 消化以凝膠純化出小片段。接著將含有編碼著殘基1-185的開放讀碼之該片段分殖到表現媒體pCEP4的Hind III和Xho I 消化體中。所得媒體，pmuOPG[l-185]編碼在位置194的離胺酸終止之截頭OPG多肽。依上述用該經轉染和藥物選擇培養過的細胞產生經調理培養基。 _-62· 本紙張尺度適用中國國家標準（CNS ) A4規格（2丨〇><297公釐了 (請先閱讀背面之注意事項再填寫本頁) 訂· 1221482 A7 B7 五、發明説明（6〇 ) 在huOPG[22-401]-Fc基因的5’端產生幾種突變，其可在殘基22至32間導入OPG的胺基酸取代，或缺失。所有突變都是因"QuickChange™部位導向突變形成套劑1’（Stratagene， San Diego, CA)以製造商建議的程序產生的。簡而言之，對含有huOPG[22-401]-Fc質體DNA模版和突變形成性引子的反應混合物用Pfu聚合酶在去氧核苷酸存在中處理之，然後依上述在熱循環器内擴增。其後取一液份的反應物經由熱震盪轉染到勝任的大腸桿菌XLl-Blue中，再予以平板培養。然後將轉形體的質體DNA定序以查證突變。下列引子配對係用來將人類OPG基因的殘基22-26缺失掉，促成huOPG[22_401]-Fc融合蛋白質的產生： 1436-11 : 5，-TGG ACC ACC CAG AAG TAC CTT CAT TAT GAC-3· (SEQ ID NO : 140) 1436-12 ： 5'.GTC ΑΤΑ ATG AAG GTA CTT CTG GGT GGT CCA-31 (SEQ ID NO : 141) 下列引子配對係用來將人類OPG基因的殘基22-28缺失掉，促成huOPG[22_401]-Fc融合蛋白質之產生：經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 1436-17 ： 5'-GGA CCA CCC AGC TTC ATT ATG ACG AAG AAA C-3* (SEQ ID NO : 142) 1436-18 ·· 5f-GTT TCT TCG TCA TAA TGA AGC TGG GTG GTC C-3' -63- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（61 ) (SEQ ID NO : 143) 下列引子配對係用來將人類OPG基因的殘基22-31缺失掉，導致lmOPG[22-401]_Fc融合蛋白質之產生： 1436-27 ： 5丨-GTG GAC CAC CCA GGA CGA AGA AAC CTC TC-3, (SEQ ID NO : 144) 1436-28 · 5f-GAG AGG TTT CTT CGT CCT GGG TGG TCC AC-3* (SEQ ID NO : 145) 下列引子配對係用來將人類OPG基因的酪胺酸28密碼子改變成***酸的，導致huOPG[22-401]-FcY28F融合蛋白質之產生： 1436-29 : 5'-CGT TTC CTC CAA AGT TCC TTC ATT ATG AC-3f (SEQ ID NO : 146) 1436-30 : 5，-GTC ATA ATG AAG GAA CTT TGG AGG AAA CG-3, (SEQ ID NO : 147) 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) _鏰· 下列引子配對係用來將人類OPG基因的脯胺酸殘基26之密碼子改變成丙胺酸的，導致huOPG[22-401]-Fc P26A融合蛋白質之產生： 1429-83 ： 5'-GGA AAC GTT TCC TGC AAA GTA CCT TCA TTA TG-3 (SEQ ID NO : 148) -64- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐) 1221482 經濟部中央標準局員工消費合作社印裝 A7 B7五、發明説明（62 ) 1429-84 : 5，-CATAATGAAGGTACTTTGCAGGAAACGTTTCC-3,（SEQJD NO : 149) 然後將每一種所得含有恰當突變的muOPG[22-401]-Fc質體轉染到人類293細胞中，依上述從調理培養基中純化出突變株OPG-Fc融合蛋白質。用實施例11中所述活體外蝕骨細胞形成檢定評估每一蛋白質的生物學活性。實施例8 大腸桿菌内的OPG表現 A.細胞表現媒體 pAMG21 從美國專利第4,710,473號中所述Amgen表現媒體系統衍生出Amgen表現媒體pCFM1656 ( ATCC #69576)，其可再轉而衍生出表現質體pAMG21。pCFM1656質體可經由下述從所述PCFM836質體（專利第4,710,473號）衍生得：（a)用T4聚合酶進行末端填充接著鈍端連接而破壞兩個内源性Nde I限制部位；（b)將含有合成PL啓動基因而在獨特Aatll與Clal限制部位之間的DNA序列用從含有PL啓動基因的pCFM636(專利第4,710,473號）所得類似片段予以取代 Aatll 5， CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT- 3f TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA- -AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA- -TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT- -TACCACTGGCGGTGATACTGAGCACAT 3f (SBQ ID NO:53) -ATGGTGACCGCCACTATGACTCGTGTAGC5r (SBQ ID NO:54) Clal -65- 本紙張尺度適用中國國家標準（CNS ) A4規格（21 OX 297公釐) ----------燊------、τ------翔 (請先閲讀背面之注意事項再填寫本頁) 1221482 A7B7 五、發明説明（63 ) 及接著（C)用下列寡核甞酸取代在獨特Cla I與Κρη I限制部位之間的小DNA序列：. 5f CGATTTGATTCTAGAAGGAGGAAT7U\CATATGGTTAACGCGTTGGAATTCGGTAC3f (SEQ ID NO：48) 3' TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5， (SEQ ID NO：49) claI Kpnl 其後可經由PCR重疊寡核苷酸突變形成和DNA序列取代進行一系列部位導引性鹼改變而從PCFM1656衍出出表現質體 pAMG21。從緊接著質體複製啓動基因PcopB的5,端之Bglll 部位（質體bp # 180)起始並朝向質體複製基因進行，其中的鹼對改變如下所列： (請先閲讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製The size of this paper is based on the National Jade Standard (CNS) A4 specification (210X297 Gongchu T 1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau Staff of the Ministry of Economic Affairs A7 B7 V. Invention Description (59) I A large restricted fragment of digestion. Use Hind III and Xho I digested the resulting plastids and gel-purified small fragments. This fragment containing the open reading code encoding 1-185 was then cloned into the expression media pCEP4 for Hind III and Xho I digestion. The resulting media, pmuOPG [l-185] Encoding a truncated OPG polypeptide terminating at the cysteine residue at position 185. Conditioned medium was generated from transfected and drug-selected cultured cells as described above. 1333-82 ·· 5 , _TCC CTT GCC CTG ACC ACT CTT _3 · (SEQ ID NO: 44) 1356-13: 5'-CCT CTG TCG ACT TAC TTT TGC GTG GCT TCT CTG TT-3, (SEQ ID NO: 45) this primer pair system Amplifies the murine OPG cDNA region encoding the amino acid 70-194 (bp 298-672) in the OPG reading and contains an artificial stop code followed by the amino acid codon (K194), followed by artificial Sal I endonuclease restriction site. The expected product contains a medium that can be used for colonization into pre-existing media Eco RI restriction site. After PCR amplification, the purified product was digested with Eco RI and Sal I endonuclease, and large fragments were purified by gel. The purified product was then cloned into the pBluescript-muOPG Fl A large restriction fragment of the Eco RI and Sal I digests of the .Fc. The resulting plastids were digested with Hind III and Xho I to gel-purify small fragments. The fragment containing the open reading sequence encoding residues 1-185 was then purified. Colonized into the Hind III and Xho I digests of the expression media pCEP4. The resulting media, pmuOPG [l-185] encodes a truncated OPG polypeptide terminated at position 194. The transfection and drug selection were used as described above. The cultured cells produce a conditioned medium. _-62 · This paper size applies the Chinese National Standard (CNS) A4 specification (2 丨〇 > < 297 mm (please read the precautions on the back before filling this page) ·· 1221482 A7 B7 V. Description of the invention (60) Several mutations are generated at the 5 'end of the huOPG [22-401] -Fc gene, which can introduce OPG amino acid substitutions between residues 22 to 32, or Deletion. All mutations are caused by " QuickChange ™ site-directed mutations 1 '(Stratagene, San Diego, CA) at the manufacturer's recommended procedures generated. In short, the reaction mixture containing the huOPG [22-401] -Fc plastid DNA template and mutation-forming primers was treated with Pfu polymerase in the presence of deoxynucleotides and then in a thermal cycler as described above. Amplification. Thereafter, a aliquot of the reaction was transfected into competent E. coli XLl-Blue via thermal shock, and plate culture was performed. The plastid DNA of the transformants was then sequenced to verify mutations. The following primer pairing lines were used to delete residues 22-26 of the human OPG gene and promote the production of the huOPG [22_401] -Fc fusion protein: 1436-11: 5, -TGG ACC ACC CAG AAG TAC CTT CAT TAT GAC-3 (SEQ ID NO: 140) 1436-12: 5 '. GTC ΑΑΑ ATG AAG GTA CTT CTG GGT GGT CCA-31 (SEQ ID NO: 141) The following primer pairs are used to use residues 22-28 of the human OPG gene The deletion caused the production of the huOPG [22_401] -Fc fusion protein: printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) 1436-17: 5'-GGA CCA CCC AGC TTC ATT ATG ACG AAG AAA C-3 * (SEQ ID NO: 142) 1436-18 ·· 5f-GTT TCT TCG TCA TAA TGA AGC TGG GTG GTC C-3 '-63- This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 V. Description of the invention (61) (SEQ ID NO: 143) The following primer pairing system was used to delete the residues 22-31 of the human OPG gene, resulting in lmOPG [22-401] Production of Fc fusion protein: 1436-27: 5 丨 -GTG GAC CAC CCA GGA CGA AGA AAC CTC TC-3, (SEQ ID NO: 144) 1436-28 · 5f-GAG AG G TTT CTT CGT CCT GGG TGG TCC AC-3 * (SEQ ID NO: 145) The following primer pairings are used to change the tyrosine 28 codon of the human OPG gene to phenylalanine, resulting in huOPG [22-401]- the fusion protein produced FcY28F: 1436-29: 5'-CGT TTC CTC CAA AGT TCC TTC ATT ATG AC-3f (SEQ ID NO: 146) 1436-30: 5, -GTC ATA ATG AAG GAA CTT TGG AGG AAA CG- 3, (SEQ ID NO: 147) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) _ 鏰 · The following primer pairs are used to remove the proline residues of the human OPG gene The codon of group 26 was changed to alanine, resulting in the production of the huOPG [22-401] -Fc P26A fusion protein: 1429-83: 5'-GGA AAC GTT TCC TGC AAA GTA CCT TCA TTA TG-3 (SEQ ID NO : 148) -64- This paper size applies Chinese National Standard (CNS) A4 (210X 297 mm) 1221482 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (62) 1429-84: 5, -CATAATGAAGGTACTTTGCAGGAAACGTTTCC-3, (SEQJD NO: 149) Then each obtained muOPG [22-401] -Fc containing the appropriate mutation The plastid was transfected into human 293 cells, and the mutant OPG-Fc fusion protein was purified from the conditioning medium as described above. The biological activity of each protein was evaluated using the in vitro osteoclast formation assay described in Example 11. Example 8 OPG expression in E. coli A. Cell expression media pAMG21 The Amgen expression media pCFM1656 (ATCC # 69576) was derived from the Amgen expression media system described in U.S. Patent No. 4,710,473, which can in turn be derived into expression plastids pAMG21. pCFM1656 plastids can be derived from the PCFM836 plastids (Patent No. 4,710,473) via: (a) end filling with T4 polymerase followed by blunt end ligation to disrupt two endogenous Nde I restriction sites; (b The DNA sequence containing the synthetic PL promoter gene and between the unique Aatll and Clal restriction sites was replaced with a similar fragment obtained from pCFM636 (patent No. 4,710,473) containing the PL promoter gene and replaced with Aatll 5, CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATATGTGTA-TGAGTA-TGAGTA-TGAGTA -TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT- -TACCACTGGCGGTGATACTGAGCACAT 3f (SBQ ID NO: 53) -ATGGTGACCGCCACTATGACTCGTGTAGC5r (SBQ ID NO: 54) Clal -65- This paper is in accordance with Chinese National Standards (CN21-XX-XX) -OX4 ----- 燊 ------, τ ------ Xiang (Please read the notes on the back before filling this page) 1221482 A7B7 V. Description of the invention (63) and then (C) use the following Oligonucleotide substitution in unique Cla I and κρη Small DNA sequence between I restriction sites: 5f CGATTTGATTCTAGAAGGAGGAAT7U \ CATATGGTTAACGCGTTGGAATTCGGTAC3f (SEQ ID NO: 48) 3 'TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5, (SEQ ID NO: 49) claI Kpnl can be mutated by PCR and then overlapped by PCR Sequence substitution made a series of site-directed base changes to derive pAMG21 from PCFM1656. Starting from the Bglll site (plastid bp # 180) at the 5, end of the plastid replication promoter PcopB and proceeding towards the plastid replication gene, the changes in the base pairs are listed below: (Please read the precautions on the back first (Fill in this page again) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs

pAMG21 bp # PCFM1656 中的bb 在PAMG21中改變成的bp # 204 T/A C/G # 428 A/T G/C # 509 G/C A/T # 617 -- ***兩G/C bp # 679 G/C T/A # 980 T/A C/G # 994 G/C A/T # 1004 A/T C/G # 1007 C/G T/A # 1028 A/T T/A # 1047 C/G T/A # 1178 G/C T/A # 1466 G/C T/A # 2028 G/C bp缺失 # 2187 C/G T/A # 2480 A/T T/A # 2499-2502 AGTG GTCA TCAC CAGT -66 · 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7B7 五、發明説明（64 # 2642pAMG21 bp # PCFM1656 bb is changed to the in PAMG21 bp # 204 T / AC / G # 428 A / TG / C # 509 G / CA / T # 617 - insert two G / C bp # 679 G / CT / A # 980 T / AC / G # 994 G / CA / T # 1004 A / TC / G # 1007 C / GT / A # 1028 A / TT / A # 1047 C / GT / A # 1178 G / CT / A # 1466 G / CT / A # 2028 G / C bp deletion # 2187 C / GT / A # 2480 A / TT / A # 2499-2502 AGTG GTCA TCAC CAGT -66 · this paper scales applicable Chinese national standard (CNS) A4 specifications (210X297 mm) 1221482 A7B7 V. Description of the invention (64 # 2642

TCCGAGC AGGCTCG 7 bp缺失 # 3435 # 3446 # 3643TCCGAGC AGGCTCG 7 bp deletion # 3435 # 3446 # 3643

c c T ///G G A T T A A/A/T/ 在獨特Aat II(pCFM1656中的位置#4364)與Sac II(在pCFM1656 中的位置#4585)限制部位之間的DNA序列係用下列DNA序列予以取代： (請先閲讀背面之注意事項再填寫本頁) 「裝· tAatll sticky end] 5· GCGTAACGTATGCATGGTCTCC- (position #4358 in pAMG21) 3· TGCACGCATTGCATACGTACCAGAGG- -CCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACT- -GGTACGCTCTCATCCCTTGACGGTCCGTAGTTTATTTTGCTTTCCGAGTCAGCTTTCTGA- -GGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGC- -CCCGGAAAGCAAAATAGACAACAAACAGCCACTTGCGAGAGGACTCATCCTGTTTAGGCG- -CGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGC- -GCCCTCGCCTAAACTTGCAACGCTTCGTTGCCGGGCCTCCCACCGCCCGTCCTGCGGGCG- -CATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGT- -GTATTTGACGGTCCGTAGTTTAATTCGTCTTCCGGTAGGACTGCCTACCGGAAAAACGCA-cc T /// GGATTAA / A / T / unique Aat II (pCFM1656 in position # 4364) and Sac II (position # 4585 in pCFM1656) restriction sites between the DNA sequences are to be substituted with the following DNA sequence: (please read the back issues of the note and then fill in this page) "installed · tAatll sticky end] 5 · GCGTAACGTATGCATGGTCTCC- (position # 4358 in pAMG21) 3 · TGCACGCATTGCATACGTACCAGAGG- -CCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACT- -GGTACGCTCTCATCCCTTGACGGTCCGTAGTTTATTTTGCTTTCCGAGTCAGCTTTCTGA- -GGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGC- -CCCGGAAAGCAAAATAGACAACAAACAGCCACTTGCGAGAGGACTCATCCTGTTTAGGCG- -CGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGC- -GCCCTCGCCTAAACTTGCAACGCTTCGTTGCCGGGCCTCCCACCGCCCGTCCTGCGGGCG- -CATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGT- -GTATTTGACGGTCCGTAGTTTAATTCGTCTTCCGGTAGGACTGCCTACCGGAAAAACGCA-

Aat 11 -TTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGGACGTCGTACTTAAC- -AAGATGTTTGAGAAAACAAATAAAAAGATTTATGTAAGTTTATACCTGCAGCATGAATTG- -TTTTAAAGTATGGGCAATCAATTGCTCCTGTTAAAATTGCTTTAGAAATACTTTGGCAGC- -AAAATTTCATACCCGTTAGTTAACGAGGACAATTTTAACGAAATCTTTATGAAACCGTCG- -GGTTTGTTGTATTGAGTTTCATTTGCGCATTGGTTAAATGGAAAGTGACCGTGCGCTTAC- -CCAAACAACATAACTCAAAGTAAACGCGTAACCAATTTACCTTTCACTGGCACGCGAATG- -TACAGCCTAATATTTTTGAAATATCCCAAGAGCTTTTTCCTTCGCATGCCCACGCTAAAC- -ATGTCGGATTATAAAAACTTTATAGGGTTCTCGAAAAAGGAAGCGTACGGGTGCGATTTG- -ATTCTTTTTCTCTTTTGGTTAAATCGTTGTTTGATTTATTATTTGCTATATTTATTTTTC- -TAAGAAAAAGAGAAAACCAATTTAGCAACAAACTAAATAATAAACGATATAAATAAAAAG- -67- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）訂經濟部中央標準局員工消費合作社印製 1221482 A7 B7 五、發明説明（65 ) -GATAATT- -CTATTAA' TTATCAACTAGAGAAGGAACAATTAATGGTATGTTCATACACGCATGTAAAAATA-TAGTTGATCTCTTCCTTGTTAATTACCATACAAGTATGTGCGTACATTTTTAT- -AACTATCTATATAGTTGTCTTTCTCTGAATGTGCAAAACTAAGCATTCCGAAGCCATTAT- -TTGATAGATATATCAACAGAAAGAGACTTACACGTTTTGATTCGTAAGGCTTCGGTAATA- -TAGCAGTATGAATAGGGAAACTAAACCCAGTGATAAGACCTGATGATTTCGCTT' -ATCGTCATACTTATCCCTTTGATTTGGGTCACTATTCTGGACTACTAAAGCGAAG- CTTTAA- .GAAATT- -TTACATTTGGAGATTTTTTATTTACAGCATTGTTTTCAAATATATTCCAATTAATCGGTG- -AATGTAAACCTCTAAAAAATAAATGTCGTAACAAAAGTTTATATAAGGTTAATTAGCCAC- -TTACTAACCTCAATCTTATTAGATGATATCCTAGTATAAAAT. TTTATTAAA' AAATAATTT, AATCGCAGTAGTA- -AATATTGCCTCCATTTTTTAGGGTAATTATCCAGAATTGAAATATCAGATTTAACCATAG- -TTATAACGGAGGTAAAAAATCCCATTAATAGGTCTTAACTTTATAGTCTAAATTGGTATC- -AATGAGGATAAATGATCGCGAGTAAATAATATTCACAATGTACCATTTTAGTCATATCAG- -TTACTCCTATTTACTAGCGCTCATTTATTATAAGTGTTACATGGTAAAATCAGTATAGTC- -ATAAGCATTGATTAATATCATTATTGCTTCTACAGGCTTTAATTTTATTAATTATTCTGT- -TATTCGTAACTAATTATAGTAATAACGAAGATGTCCGAAATTAAAATAATTAATAAGACA- -AAGTGTCGTCGGCATTTATGTCTTTCATACCCATCTCTTTATCCTTACCTATTGTTTGTC- -TTCACAGCAGCCGTAAATACAGAAAGTATGGGTAGAGAAATAGGAATGGATAACAAACAG- (請先閲讀背面之注意事項再填寫本頁) 、?7 經濟部中央標準局員工消費合作社印製 -GCAAGTTTTGCGTGTTATATATCATTAAAACGGTAATAGATTGACATTTGATTCTAATAA- -CGTTCAAAACGCACAATATATAGTAATTTTGCCATTATCTAACTGTAAACTAAGATTATT--ATTGGATTTTTGTCACACTATTATATCGCTTGAAATACAATTGTTTAACATAAGTACCTG- -TAACCTAAAAACAGTGTGATAATATAGCGAACTTTATGTTAACAAATTGTATTCATGGAC--TAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGATTAATCGATTTGATT- -ATCCTAGCATGTCCAAATGCGTTCTTTTACCAAACAATATCAGCTAATTAGCTAAAdTAA- -CTAGATTTGTTTTAACTAATTAAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGA- -GATCTAAACAAAATTGATTAATTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCT-Aat 11 -TTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGGACGTCGTACTTAAC- -AAGATGTTTGAGAAAACAAATAAAAAGATTTATGTAAGTTTATACCTGCAGCATGAATTG- -TTTTAAAGTATGGGCAATCAATTGCTCCTGTTAAAATTGCTTTAGAAATACTTTGGCAGC- -AAAATTTCATACCCGTTAGTTAACGAGGACAATTTTAACGAAATCTTTATGAAACCGTCG- -GGTTTGTTGTATTGAGTTTCATTTGCGCATTGGTTAAATGGAAAGTGACCGTGCGCTTAC- -CCAAACAACATAACTCAAAGTAAACGCGTAACCAATTTACCTTTCACTGGCACGCGAATG- -TACAGCCTAATATTTTTGAAATATCCCAAGAGCTTTTTCCTTCGCATGCCCACGCTAAAC- -ATGTCGGATTATAAAAACTTTATAGGGTTCTCGAAAAAGGAAGCGTACGGGTGCGATTTG- -ATTCTTTTTCTCTTTTGGTTAAATCGTTGTTTGATTTATTATTTGCTATATTTATTTTTC- -TAAGAAAAAGAGAAAACCAATTTAGCAACAAACTAAATAATAAACGATATAAATAAAAAG- -67- This paper scales applicable Chinese National Standard (CNS) A4 size (210X297 public (%) Order printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 1221422 A7 B7 V. Description of Invention (65) -GATAATT- -CTATTAA 'TTATCAACTAGAGAAGGAACAATTAATGGTATGTTCATACACGCATGTAAAAATA-TAGTTGATCTCTTCCTTGTCTCCATCACTAGAGTGTGCGTACATTTTTATCTACTATCTAT CATTAT- -TTGATAGATATATCAACAGAAAGAGACTTACACGTTTTGATTCGTAAGGCTTCGGTAATA- -TAGCAGTATGAATAGGGAAACTAAACCCAGTGATAAGACCTGATGATTTCGCTT '. -ATCGTCATACTTATCCCTTTGATTTGGGTCACTATTCTGGACTACTAAAGCGAAG- CTTTAA- .GAAATT- -TTACATTTGGAGATTTTTTATTTACAGCATTGTTTTCAAATATATTCCAATTAATCGGTG- -AATGTAAACCTCTAAAAAATAAATGTCGTAACAAAAGTTTATATAAGGTTAATTAGCCAC- -TTACTAACCTCAATCTTATTAGATGATATCCTAGTATAAAAT TTTATTAAA' AAATAATTT, AATCGCAGTAGTA- -AATATTGCCTCCATTTTTTAGGGTAATTATCCAGAATTGAAATATCAGATTTAACCATAG- -TTATAACGGAGGTAAAAAATCCCATTAATAGGTCTTAACTTTATAGTCTAAATTGGTATC- -AATGAGGATAAATGATCGCGAGTAAATAATATTCACAATGTACCATTTTAGTCATATCAG- -TTACTCCTATTTACTAGCGCTCATTTATTATAAGTGTTACATGGTAAAATCAGTATAGTC- -ATAAGCATTGATTAATATCATTATTGCTTCTACAGGCTTTAATTTTATTAATTATTCTGT- -TATTCGTAACTAATTATAGTAATAACGAAGATGTCCGAAATTAAAATAATTAATAAGACA- - AAGTGTCGTCGGCATTTATGTCTTTCATACCCATCTCTTTATCCTTACCTATTGTTTGTC- -TTCACAGCAGCCGTAAATACAGAAAGTATGGGTAGAGAAATAGGAATGGATAACAAACAG- (Please read the notes on the back before filling out this page),? 7 Central Standards Bureau of the Ministry of Economic Affairs Consumer cooperatives printed -GCAAGTTTTGCGTGTTATATATCATTAAAACGGTAATAGATTGACATTTGATTCTAATAA- -CGTTCAAAACGCACAATATATAGTAATTTTGCCATTATCTAACTGTAAACTAAGATTATT - ATTGGATTTTTGTCACACTATTATATCGCTTGAAATACAATTGTTTAACATAAGTACCTG- -TAACCTAAAAACAGTGTGATAATATAGCGAACTTTATGTTAACAAATTGTATTCATGGAC - TAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGATTAATCGATTTGATT- -ATCCTAGCATGTCCAAATGCGTTCTTTTACCAAACAATATCAGCTAATTAGCTAAAdTAA- -CTAGATTTGTTTTAACTAATTAAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGA- -GATCTAAACAAAATTGATTAATTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCT-

SacII -GCTCACTAGTGTCGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAA- -CGAGTGATCACAGCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTT- -GAAGAAGAAGAAGAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATA- -CTTCTTCTTCTTCTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTAT- -ACTAGCATAACCCCTTGGGGCCTCT7VAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGG- -TGATCGTATTGGGGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCC- -AACCGCTCTTCACGCTCTTCACGC 31 [SacII sticky end] (SBQ ID NO：50) -TTGGCGAGAAGTGCGAGAAGTG 5· (position #5904 in pAMG21) (SBQ ID NO：46) 68 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（66 )SacII -GCTCACTAGTGTCGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAA- -CGAGTGATCACAGCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTT- -GAAGAAGAAGAAGAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATA- -CTTCTTCTTCTTCTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTAT- -ACTAGCATAACCCCTTGGGGCCTCT7VAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGG- -TGATCGTATTGGGGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCC- -AACCGCTCTTCACGCTCTTCACGC 31 [SacII sticky end] (SBQ ID NO: 50) -TTGGCGAGAAGTGCGAGAAGTG 5 · (position # 5904 in pAMG21) (SBQ ID NO: 46) 68 This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 V. Description of invention (66)

於此取代DNA序列黏狀末端的連接中，外部的八肘II和Sac II 部位即被破壞。在經取代DNA中有獨特的Aat π和Sac II部位。 · nAMG22-His 表現質體pAMG22-His可以經由將pAMG22中在獨特Nde I (#4795)與EcoRI(#4818)限制部位之間的小DNA序列用下列寡核甞酸複體取代而從Amgen表現媒體pAMG22衍生出：In this connection that replaces the sticky ends of the DNA sequence, the outer eight elbow II and Sac II sites are destroyed. There are unique Aat π and Sac II sites in the substituted DNA. NAMG22-His expression pAMG22-His can be expressed from Amgen by substituting the small DNA sequence between the unique Nde I (# 4795) and EcoRI (# 4818) restriction sites in pAMG22 with the following oligonucleotide complex Media pAMG22 derived:

Ndel Nhel _ EcoRI 5! TATGAAACATCATCACCATCACCATCATGCTAGCGTTAACGCGTTGG 31 (SBQ ID N0：51) 3· ACTTTGTAGTAGTGGTAGTGGTAGTACGATCGCAATTGCGCAACCTTAA 5' (SBQ ID NO:52)Ndel Nhel _ EcoRI 5! TATGAAACATCATCACCATCACCATCATGCTAGCGTTAACGCGTTGG 31 (SBQ ID N0: 51) 3. ACTTTGTAGTAGTGGTAGTGGTAGTACGATCGCAATTGCGCAACCTTAA 5 '(SBQ ID NO: 52)

MetLysHisHisHisHisHisHisHisAlaSerValAsnAlaLeuGlu (SEQ ID NO： 168) PAMG22 從美國專利第4,710,473號（1987年12月1日授與）所述Amgen 表現媒體系統可衍生出Amgen表現媒體pCFM1656(ATCC #69576)，其轉而可衍生表現質體pAMG22。質體pCFM1656 可經由下述而從所述質體PCFM836(專利第4,710,473號）衍生出： (a)經由用T4聚合酶末端填充接著鈍端連接而破壞兩個内源性Ndel限制部位；（b)將在獨特Aat II與Clal限制部位之間含有合成PL啓動基因的DNA序列用得自pCFM636(專利第 4,710,473號）含有PL啓動基因的類似片段予以取代MetLysHisHisHisHisHisHisHisHisAslaSerValAsnAlaLeuGlu (SEQ ID NO: 168) PAMG22 The Amgen Performance Media System described in US Patent No. 4,710,473 (granted December 1, 1987) can be derived from Amgen Performance Media pCFM1656 (ATCC # 69576), which in turn can be derived Plastid pAMG22. Plastid pCFM1656 can be derived from said plastid PCFM836 (Patent No. 4,710,473) by: (a) destroying two endogenous Ndel restriction sites by filling with T4 polymerase end followed by blunt end attachment; (b ) The DNA sequence containing the synthetic PL promoter gene between the unique Aat II and Clal restriction sites was replaced with a similar fragment containing the PL promoter gene from pCFM636 (Patent No. 4,710,473)

Aat II 5f CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT-3f TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA- -AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA- -TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT- -TACCACTGGCGGTGATACTGAGCACAT 3/ (SBQ ID NO:53) -ATGGTGACCGCCACTATGACTCGTGTAGC5f (SBQ ID NO:54)Aat II 5f CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT-3f TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA- -AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA- -TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT- -TACCACTGGCGGTGATACTGAGCACAT 3 / (SBQ ID NO: 53) -ATGGTGACCGCCACTATGACTCGTGTAGC5f (SBQ ID NO: 54)

Clal ^^尺度適用中國國家標準（CNS ) A4規格（210X297公釐1 (請先聞讀背面之注意事項再填寫本頁) •裝· 、11 經濟部中央標準局員工消費合作社印製 -69- _ 1221482 列者： dAMG22 bn# A7 B7 五、發明説明（67 ) 及接著（C)用下列寡核苷酸取代在獨特Cla I與Κρη I限制部位之間的小DNA序列： 5f CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3f(SE)Q ID NO：55) 3， TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5r (SBQ ID NO： 56) Clal KpnlClal ^^ standard is applicable to China National Standard (CNS) A4 specifications (210X297mm1 (please read the precautions on the back before filling out this page) _ 1221482 Listed by: dAMG22 bn # A7 B7 5. Description of the invention (67) and then (C) Replace the small DNA sequence between the unique Cla I and κρη I restriction site with the following oligonucleotide: 5f CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3f (SE ) Q ID NO: 55) 3, TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5r (SBQ ID NO: 56) Clal Kpnl

然後可從pCFM1656經由PCR重疊寡核苷酸突變形成和DNA 序列取代進行一系列部位導引性鹼變換而衍生出表現質體 pAMG22。從緊接在質體複製啓動基因PcopB 5’端的Bgl II部位起始並朝向質體複製基因進行時，其鹼對變換爲如下所在DCFM1656中的bb在pAMG22中改變成的bp (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 # # # # # # # # # # # # # # # # 4 4 7 8 7 8 9790400247 0201789000014 6 9 1 1 11 11 11 11 6 6 2028 2187 2480 # 2499-2502 # 2642Then overlapping oligonucleotides via PCR from pCFM1656 mutagenesis and DNA sequence substitutions series of guide portions of the base transformation derived expression plasmid pAMG22. From the immediately plastid replication initiation gene PcopB 5 'end toward the Bgl II site of plasmid replication initiation gene, which base is located below the transformation into the DCFM1656 bb changed to in pAMG22 in BP (back surface Read Please pay attention to this page and fill in this page) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs # # # # # # # # # # # # # # # # 4 4 7 8 7 8 9790400247 0201789000014 6 9 1 1 11 11 11 11 66202821872480 # 2499-2502 # 2642

T/A A/T G/C G/C T/A G/C A/T C/G A/T C/G G/C G/C G/C C/G A/T AGTG TCAC TCCGAGC C/G G/C A/T ***兩G/C bp T/A C/G A/T C/G T/A T/A T/A T/A T/A bp缺失 T/A T/A GTCA CAGT 7 bp缺失 -70- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X297公釐） 1221482 A7 B7 五、發明説明（68 )T / AA / TG / CG / CT / AG / CA / TC / GA / TC / GG / CG / CG / CC / GA / T AGTG TCAC TCCGAGC C / GG / CA / T Insert two G / C bp T / AC / GA / TC / GT / AT / AT / AT / AT / A bp missing T / AT / A GTCA CAGT 7 bp missing -70- This paper size applies to China National Standard (CNS) A4 specification (21〇297mm) 1221482 A7 B7 V. Description of the invention (68)

AGGCTCG # 3435 # 3446 # 3643 ξ/τ 用下列DNA序列取代在獨特Aat II(pCFM1656中的位置#4364) 與Sac II(pCFM1656中的位置#4585)限制部位之間的DNA序列： [Aatll sticky end] (position #4358 in pAMG22) 51 GCGTAACGTATGCATGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAA- 3' TGCACGCATTGCATACGTACCAGAGGGGTACGCTCTCATCCCTTGACGGTCCGTAGTT- -ATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG- -TATTTTGCTTTCCGAGTCAGCTTTCTGACCCGGAAAGCAAAATAGACAACAAACAGCCAC- -AACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGG- -TTGCGAGAGGACTCATCCTGTTTAGGCGGCCCTCGCCTAAACTTGCAACGCTTCGTTGCC- -CCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAG- -GGGCCTCCCACCGCCCGTCCTGCGGGCGGTATTTGACGGTCCGTAGTTTAATTCGTCTTC- -GCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAAT- -CGGTAGGACTGCCTACCGGAAAAACGCAAAGATGTTTGAGAAAACAAATAAAAAGATTTA-AGGCTCG # 3435 # 3446 # 3643 ξ / τ Replace the DNA sequence between the unique Aat II (position # 4364 in pCFM1656) and Sac II (position # 4585 in pCFM1656) restriction sites with the following DNA sequence: [Aatll sticky end ] (position # 4358 in pAMG22) 51 GCGTAACGTATGCATGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAA- 3 'TGCACGCATTGCATACGTACCAGAGGGGTACGCTCTCATCCCTTGACGGTCCGTAGTT- -ATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG- -TATTTTGCTTTCCGAGTCAGCTTTCTGACCCGGAAAGCAAAATAGACAACAAACAGCCAC- -AACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGG- -TTGCGAGAGGACTCATCCTGTTTAGGCGGCCCTCGCCTAAACTTGCAACGCTTCGTTGCC- -CCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAG- -GGGCCTCCCACCGCCCGTCCTGCGGGCGGTATTTGACGGTCCGTAGTTTAATTCGTCTTC- -GCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAAT- -CGGTAGGACTGCCTACCGGAAAAACGCAAAGATGTTTGAGAAAACAAATAAAAAGATTTA-

Aat 11 -ACATTCAAATATGGACGTCTCATAATTTTTAAAAAATTCATTTGACAAATGCT,Aat 11 -ACATTCAAATATGGACGTCTCATAATTTTTAAAAAATTCATTTGACAAATGCT,

-TGTAAGTTTATACCTGCAGAGTATTAAAAATTTTTTAAGTAAACTGTTTACGAT AAAATTC- TTTTAAG- (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 -TTGATTAATATTCTCAATTGTGAGCGCTCACAATTTATCGATTTGATTCTAGATTTGTTT- -AACTAATTATAAGAGTTAACACTCGCGAGTGTTAAATAGCTAAACT7VAGATCTAAACTCA- -TAACTAATTAAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGAGCTCACTAGTGT- -ATTGATTAATTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCTCGAGTGATCACA- SacII -CGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAAGAAGAAGAAGAA 垂 -GCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTTCTTCTTCTTCTT- -GAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACC- -CTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTATTGATCGTATTGG- -CCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACCGCTCTTCA- -GGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCCTTGGCGAGAAGT- -CGCTCTTCACGC 3· (SBQ ID NO:58) -GCGAGAAGTG 5， {SBQ ID NO :57) [SacII sticky end] (position #5024 in pAMG22) -71 - -縐- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（69 ) 於此取代DNA序列所含黏狀末端的連接中，外側Aat II和Sac II部位都被破壞。而在取代DNA中含有獨特Aat II和Sac II部位。 B·人類 OPG Met『32-4011 於此實施例中，所用表現媒體的pAMG21，其爲pCFM1656 (ATCC登錄號碼69576)的衍生物，其含有恰當的限制部位供 lux PR啓動基因下游的基因***所用。（參看美國專利第 5，169,318號有關Iux^l現系統的説明）。所用的宿主細胞爲 GM120(ATCC登錄號碼55764)。此宿主具有整合到原養型大腸桿菌K12宿主的宿主染色體中所含第二部位内的lacIQ啓動基因和lacl基因。其他常用的大腸桿菌表現媒體和宿主細胞也適用來表現。經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 依下述將編碼人類OPG多肽的N-端甲硫胺酸和胺基酸32-401之DNA序列置於質體表現媒體pAMG21中的luxPR啓動基因之控制下。要完成此舉，使用含有人類OPG cDNA的質體 pRcCMV-Hu OPG DNA作爲模版且用寡核苷酸#1257-20和 #1257-19作爲引子進行PCR，其中包括30個循環的熱循環，每一循環爲：94°C 20秒，接著37°C 30秒，接著72°C 30秒。將所得PCR樣品在瓊脂糖凝膠上解析，將PCR產物剪開，純化，並用Κρη I和BamHI核酸内切限制酶予以限制消化再純化。將合成寡核苷酸#1257-21和#1257-22分別用T4聚核甞酸激酶和ATP予以磷酸化後，將其混合在一起，於94°C下加熱並使其慢慢冷卻到室溫以形成含有Nde I和Κρη I黏狀末端的寡核甞酸聯結子複體。將在寡核苷酸#1257-21與# 1257-22之間形成且含有Nde I和Κρη I粘性末端的磷酸化聯結子複體及 -72- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） 1221482 A7 __B7 五、發明説明（70 ) 用寡核苷酸引子#1257-20和#1257-19所產生且經ΚρηΙ和BamHI 消化並純化過的PCR產物（參看上述）經由使用標準重組DNA 方法導引地插置在質體媒體PAMG21的兩個部位，亦即Nde I 部位與BamHI部位之間（參看圖14A及下面的序列）。合成聯結子係利用大腸桿菌密碼子且提供一個N-端甲硫胺酸。選擇培養出兩株系並分離出質體DNA，且於隨後經由DNA 序列確定人類OPG***體。所得含有在架構内緊接在甲硫胺酸之後的人類OPG多肽所含胺基酸32-401之質體pAMG21稱爲 pAMG21-huOPG met[32-401]或 pAMG21-huOPG met[32-401]。 01igo#1257-19 5丨_TACGCACTGGATCCnATAAGCAGCITATmTACTGATrGGAC>3.(SEQ ID NO : 59) Oligo#1257-20 5,-GTCCTCCTGGTACCTACCTAAAACAAC-3, (SEQ ID NO ： 60) 01igo#1257-21 5，-TATGGATGAAGAAACTTCTCATCAGCTGCTGTGTGATAAATGTCC GCCGGGTAC-3,（SEQ ID NO ·· 61) 01igo#1257-22 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 5，-CCGGCGGACATTTATCACACAGCAGCTGATGAGAAGTTTCTTCATC CA-3，(SEQ ID NO : 47) pAMG21-huOPG met[32_401]在大腸桿菌 GM_120 内於含有 20微克/毫升卡那黴素的2XYT培養基中的培養物係在謗導之前置於30°C下溫置。在添加合成自動謗導劑N-(3-氧基已醯基）-DL-同絲胺酸内酯到培養基中達3 0毫微克/毫升的最 _-73- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X 297公釐了 1221482 經濟部中央標準局員工消費合作社印製 A7 __B7___ 五、發明説明（71 ) 後濃度且將培養物置於30°C或37°C下培育6小時後即達到 huOPG met[32-401]基因產物表現之謗導。於6小時後，以顯微術檢查細菌培養物中的包涵體之存在，接著予以離心成丸。在經謗導的培養物中觀察到折射光的包涵體，顯示在大腸桿菌中產生某些不溶性重組huOPG met[32-401]基因產物。將一些細菌沈著丸再懸浮於10 mM Tris-HCl/ pH 8，1 mM EDTA之中並添加2X Laemlli樣品緩衝液到1 X最後濃度和-氫硫基乙醇到5 %最後濃度而予以直接溶裂，再以 SDS-PAGE分析。在含有30°C和37°C謗導培養物的總細胞溶裂物之SDS-PAGE凝膠上觀察到在約42 kDa處比在第2道上 30°C未經謗導培養物所得總細胞溶裂物實質地更強的考馬斯 (coomassie)染色帶（圖14B)。預期的基因產物爲具有370胺基酸長度及約42.2 kDa預期分子量者。在37°C下謗導6小時後，將另一培養物沈著成丸，再施以包涵體分離（參看下文）或以微流體化處理。將要用微流體化處理的丸再懸浮於25 mM Tris-HCl/pH 8，0.5 M NaCl 緩衝液中並通過 Microfluidizer Model 1108(Microfluidics Corp.)20次後收集起來。於收集所得樣品（微流體化總溶裂物）中取出一液份，並將剩餘物以20,000 X g離心2 0分鐘沈著成丸。取出離心後的上澄液（微流體化可溶部份）並將沈丸重新懸浮在25 mM Tris-HCl/pH 8，0.5 M NaCl，6M脲溶液中（微流體化不溶部份）。於一液份總可溶部份或不溶部份中加入等體積的2X Laemalli樣品緩衝液和卢·氫硫基乙醇到5 %最後濃度。然後以SDS-PAGE分析該等樣品。於不溶部份中發現明顯量的重 •74- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閱讀背面之注意事項再填寫本頁)-TGTAAGTTTATACCTGCAGAGTATTAAAAATTTTTTAAGTAAACTGTTTACGAT AAAATTC- TTTTAAG- (Please read the Notes on the back to fill out this page) Ministry of Economic Affairs Bureau of Standards employees consumer cooperatives printed -TTGATTAATATTCTCAATTGTGAGCGCTCACAATTTATCGATTTGATTCTAGATTTGTTT- -AACTAATTATAAGAGTTAACACTCGCGAGTGTTAAATAGCTAAACT7VAGATCTAAACTCA- -TAACTAATTAAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGAGCTCACTAGTGT- -ATTGATTAATTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCTCGAGTGATCACA- SacII -CGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAAGAAGAAGAAGAA down -GCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTTCTTCTTCTTCTT- -GAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACC - -CTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTATTGATCGTATTGG- -CCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACCGCTCTTCA- -GGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCCTTGGCGAGAAGT- -CGCTCTTCACGC 3 · (SBQ ID NO: 58) -GCGAGAAGTG 5, {SBQ ID NO: 57) [SacII sticky end] (position # 5024 in pAMG22) -71 - - crepe - This paper size applies to China National Standard (CNS) Α4 specifications 210X 297 mm) 1221482 A7 B7 V. invention is described in (69) connected thereto a DNA sequence contained in the substituted sticky and blunt ends, the outside Aat II and Sac II sites are destroyed. The replacement DNA contains unique Aat II and Sac II sites. B. Human OPG Met "32-4011 In this example, pAMG21, the expression medium used, is a derivative of pCFM1656 (ATCC accession number 69576), which contains appropriate restriction sites for gene insertion downstream of the lux PR promoter gene . (See U.S. Patent No. 5,169,318 for a description of the current Iux ^ l system). The host cell used was GM120 (ATCC accession number 55764). This host has a lacIQ promoter gene and a lacl gene integrated into the second site contained in the host chromosome of the prototrophic E. coli K12 host. Other commonly used E. coli expression media and host cells are also suitable for expression. Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). Place the DNA sequences of the N-terminal methionine and amino acid 32-401 encoding human OPG polypeptide as follows Under the control of the luxPR promoter in the plastid expression media pAMG21. To do this, PCR was performed using pRcCMV-Hu OPG DNA containing human OPG cDNA as a template and oligonucleotides # 1257-20 and # 1257-19 as primers, which included 30 cycles of thermal cycling, each One cycle is: 94 ° C for 20 seconds, then 37 ° C for 30 seconds, and then 72 ° C for 30 seconds. The obtained PCR sample was analyzed on an agarose gel, and the PCR product was cut open, purified, and subjected to restriction digestion and purification with Kpη I and BamHI endonucleases. The synthetic oligonucleotides # 1257-21 and # 1257-22 were phosphorylated with T4 polynucleotide kinase and ATP, respectively, mixed together, heated at 94 ° C and allowed to slowly cool to room temperature. Warm to form oligonucleotide complexes containing Nde I and Kρη I sticky ends. Phosphorylated linker complex formed between oligonucleotides # 1257-21 and # 1257-22 and containing Nde I and κρη I sticky ends and -72- This paper is in accordance with Chinese National Standard (CNS) Α4 specifications (210 × 297 mm) 1221482 A7 __B7 V. Description of the invention (70) The PCR products (see above) produced by oligonucleotide primers # 1257-20 and # 1257-19 and digested and purified by κρηΙ and BamHI are used. The standard recombinant DNA method is inserted between two parts of the plastid media PAMG21, namely, between the Nde I site and the BamHI site (see Figure 14A and the sequence below). The synthetic linker system uses E. coli codons and provides an N-terminal methionine. Two lines were selected and cultured, and plastid DNA was isolated, and then a human OPG insert was determined by the DNA sequence. The resulting pAMG21 containing the amino acid 32-401 of the human OPG polypeptide immediately behind methionine in the framework is called pAMG21-huOPG met [32-401] or pAMG21-huOPG met [32-401] . 01igo # 1257-19 5 丨 _TACGCACTGGATCCnATAAGCAGCITATmTACTGATrGGAC > 3. (SEQ ID NO: 59) Oligo # 1257-20 5, -GTCCTCCTGGTACCTACCTAAAACAAC-3, (SEQ ID NO: 60) 01igo # 1257-21 5, -TATGGATGAAGAAACTTCTCATCAGGTAGAGGTACAGCT (SEQ ID NO ·· 61) 01igo # 1257-22 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) 5, -CCGGCGGACATTTATCACACAGCAGCTGATGAGAAGTTTCTTCATC CA-3, (SEQ ID NO: 47) The culture of pAMG21-huOPG met [32_401] in E. coli GM_120 in 2XYT medium containing 20 μg / ml kanamycin was warmed at 30 ° C before inducing. Add the synthetic auto-defense agent N- (3-oxyhexyl) -DL-isoselide to the culture medium up to 30 nanograms / ml. _-73- This paper size applies to Chinese national standards (CNS) A4 specification (21〇X 297 mm 1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 __B7___ V. Description of the invention (71) After concentration and culture at 30 ° C or 37 ° C 6 After hours, the expression of the huOPG met [32-401] gene product was reached. After 6 hours, the presence of inclusion bodies in the bacterial culture was examined microscopically, and then centrifuged into pellets. Refracted light inclusions were observed in the body, showing that some insoluble recombinant huOPG met [32-401] gene products were produced in E. coli. Some bacterial pellets were resuspended in 10 mM Tris-HCl / pH 8, 1 mM EDTA Add 2X Laemlli sample buffer to the final concentration of 1 X and -hydrothioethanol to the final concentration of 5% and directly lyse it, and then analyze it by SDS-PAGE. Culture at 30 ° C and 37 ° C. The total lysate of the product was observed on the SDS-PAGE gel at about 42 kDa than in lane 2. Coomassie staining bands (Figure 14B) with substantially stronger total lysates from uncultured cultures at 30 ° C. The expected gene product is a 370 amino acid length and an expected molecular weight of approximately 42.2 kDa After 6 hours of incubation at 37 ° C, another culture was pelleted, and then the inclusion body was separated (see below) or treated with microfluidization. The microfluidized pellet was resuspended in 25 mM Tris-HCl / pH 8,0.5 M NaCl buffer and collected 20 times through microfluidizer Model 1108 (microfluidics Corp.). the resulting samples were removed for collecting (microfluidized total lysates) parts of a solution, Centrifuge the residue at 20,000 X g for 20 minutes to pellet. Remove the centrifuged supernatant (microfluidized soluble fraction) and resuspend the pellet in 25 mM Tris-HCl / pH 8, 0.5 M NaCl, 6M urea solution (microfluidized insoluble part). Add an equal volume of 2X Laemalli sample buffer and Lu-Hydroxythioethanol to a final concentration of 5% in one part of the total soluble or insoluble part. The samples were then analyzed by SDS-PAGE. Significant weight • 74- This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm) (Please read the precautions on the back before filling this page)

、1T, 1T

Ur- 1221482 A7 B7 五、發明説明（72 ) 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 組huOPG met[32-401]基因產物。要純化出該重組蛋白質時，係依下文所述先純化過包涵體：用在Beckman J-6B離心機中所裝的JS-4.2轉子以4,900 xg在4°C進行密度梯度離子 1 5分鐘而從培養基分離出細菌細胞。將細菌沈丸再懸浮於5 毫升水中後，用水稀釋到10毫升最後體積。將此懸浮液轉移到已在冰中冷卻過的不銹鋼杯中並用裝備著標準尖頭的 Branson Sonifier施以聲波碎裂（動力設定=5，負載循環 =95%，80次破裂）。將聲波處理過的細胞懸浮液置於 Beckman Optima TLX超離心機中的TLA 100.3轉子内以 195,000 X g在23°C下離心5到10分鐘。丟棄上澄液並用注射瓶以水流沖洗沈丸。用微調刀刮取收集沈丸並轉移到玻璃句化器（15毫升容量）。於勻化器中加入5毫升的Percoll溶液 (75%液體Percoll，0.15M氣化鈉）並將内容物勻化到均勻地懸浮爲止。經由添加Percoll溶液使體積增加到19.5毫升，混合，並分配到3個Beckman Quick-Seal管（13x32毫米）之中。根據製造商的説明將諸管密封。將該等管置於Beckman TLA 100.3 轉子内於 23°C下，以 20,000 rpm(21，600 xg)離子 30 分鐘。檢驗各管的恰當分帶圖案。要回收折光體時，係經回收梯度部份並集中起來後，用水稀釋。離心沈著包涵體，並於SDS-PAGE後估測蛋白質濃度。取一份依下述分離出的包涵體溶解在含有5%卢-氫硫基乙醇的IX Laemlli樣品中並在SDS-PAGE凝膠上解析，該分離出的包涵體可提供高度純化的重組huOPG[32-401]基因產物。將在SDS-聚丙烯醯胺凝膠上解析出包涵體之後可觀察 -75- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（73 ) 到的主要〜42 kDa帶從個別凝膠上剪下並基本上依 Matsudaira et al. J. Biol. Chem. 262· 10-35(1987)中所述測定 N-端胺基酸序列。於1 9循環後定出下列序列： NH2-MDEETASHQLLCDKCPPGTY-COOH (SEQ ID NO ： 62) 此序列經發現與經由細菌表現媒體提供甲硫胺酸所產生的卩八1^1〇211111-〇?〇11^对32-401]表現媒體所編碼之前面19個胺基酸相同。 C.人類 OPG meU22-4011 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 將編碼人類OPG的N-端甲硫胺酸和胺基酸22至401之DNA 序列依下文所述置於原核生物質體表現媒體pAMG21中的 luxPR啓動基因的控制之下。將分離出的pAMG21-huOPG met[32-401]質體DNA(參看B段）用Κρη I和BamH I核酸内切限制酶切劈並將所得片段置於瓊脂糖凝膠上分離。用標準方法從凝膠上分離出B片段（〜1064 bp片段）。用B段中所述方法將合成寡核苷酸（Oligos) #1267·06和1267-07分別磷酸化並使其形成寡核替酸聯結子複體，其含有Nde I和Κρη I黏性末端。該合成聯結子複體係利用大腸样菌密碼子並提供Ν-端甲硫胺酸。將含有Nde I和Κρη I黏狀末端的磷酸化寡核甞酸聯結子與分離出的經Κρη I和BamHI核酸内切限制酶消化過的pAMG21-huOPG met[32-401]所含〜1064 bp片段經由使用標準重組DNA方法導引地插置在pAMG21的Nade I與BamHI 部位之間。將連接混合物以electroporation利用製造商的程序轉形到大腸桿菌宿主393内。選擇出菌株，分離出質體 DNA，並實施DNA定序以查證huOPG met[22-401]基因的 -76- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（74 ) DNA序列。Ur-1221482 A7 B7 V. Description of Invention (72) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) Group huOPG met [32-401] gene product. To purify the recombinant protein, the inclusion bodies were first purified as follows: using a JS-4.2 rotor mounted in a Beckman J-6B centrifuge at 4,900 xg at 4 ° C for 15 minutes at a density gradient Bacterial cells were isolated from the culture medium. After resuspending the pellet of bacteria in 5 ml of water, it was diluted with water to a final volume of 10 ml. Transfer this suspension to a stainless steel cup that has been cooled in ice and sonically break with a Branson Sonifier equipped with a standard tip (power setting = 5, load cycle = 95%, 80 bursts). The sonicated cell suspension was centrifuged in a TLA 100.3 rotor in a Beckman Optima TLX ultracentrifuge at 195,000 X g for 5 to 10 minutes at 23 ° C. Discard the clear solution and rinse the pellet with water from the injection bottle. Collect the pellets by scraping with a fine adjustment knife and transfer to a glass sentencer (15 ml capacity). Add 5 ml of Percoll solution (75% liquid Percoll, 0.15M sodium gaseous solution) to the homogenizer and homogenize the contents until it is suspended uniformly. Increase the volume to 19.5 ml by adding Percoll solution, mix, and dispense into 3 Beckman Quick-Seal tubes (13x32 mm). The tubes were sealed according to the manufacturer's instructions. Place the tubes in a Beckman TLA 100.3 rotor at 23 ° C for 30 minutes at 20,000 rpm (21,600 xg). Check the proper striping pattern of each tube. To recover the refractories, the gradient part is collected and concentrated, and then diluted with water. The inclusion bodies were pelleted by centrifugation, and the protein concentration was estimated after SDS-PAGE. Take an aliquot of the isolated inclusion bodies dissolved in a IX Laemlli sample containing 5% lu-hydrothioethanol and analyze on an SDS-PAGE gel. The isolated inclusion bodies can provide highly purified recombinant huOPG [32-401] Gene products. Observables will be observed after the inclusion bodies are resolved on the SDS-polyacrylamide gel. -75- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 A7 B7 V. Description of the invention (73) to The main ~ 42 kDa band was cut from individual gels and the N-terminal amino acid sequence was determined essentially as described in Matsudaira et al. J. Biol. Chem. 262 · 10-35 (1987). After 19 cycles, the following sequence was determined: NH2-MDEETASHQLLCDKCPPGTY-COOH (SEQ ID NO: 62) This sequence was discovered and produced by the supply of methionine via a bacterial expression media. 1 ^ 1 10211111-〇? 〇 11 ^ pairs 32-401] The previous 19 amino acids encoded by the performance media are the same. C. Human OPG meU22-4011 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) The N-terminal methionine and amino acids 22 to 401 of human OPG will be encoded The DNA sequence was placed under the control of the luxPR promoter in the prokaryotic biomass expression media pAMG21 as described below. The isolated pAMG21-huOPG met [32-401] plastid DNA (see paragraph B) was cleaved with κρη I and BamH I endonuclease restriction enzymes and the resulting fragment was separated on an agarose gel. The B fragment (~ 1064 bp fragment) was isolated from the gel using standard methods. The synthetic oligonucleotides (Oligos) # 1267 · 06 and 1267-07 were phosphorylated and formed into oligonucleotide complexes containing Nde I and κρη I sticky ends, respectively, as described in paragraph B. . The synthetic linker complex utilizes coliform codons and provides N-terminal methionine. Contains ~ 1064 bp of phosphorylated oligonucleotide linkers containing Nde I and κρη I sticky ends and isolated pAMG21-huOPG met [32-401] digested with κρη I and BamHI endonuclease The fragment was inserted between the Nade I and BamHI sites of pAMG21 via a guide using standard recombinant DNA methods. The ligation mixture was transformed into E. coli host 393 by electroporation using the manufacturer's procedure. Select the strain, isolate the plastid DNA, and perform DNA sequencing to verify the -76- of the huOPG met [22-401] gene. This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 A7 B7 5. Description of the invention (74) DNA sequence.

Oligo #1267-06 5f-TAT GGA AAC TTT TCC TCC AAA ATA TCT TCA TTA TGA TGA AGA AAC TTC TCA TCA GCT GCT GTG TGA TAA ATG TCC GCC GGG TAC-3· (SEQ ID NO ·· 63)Oligo # 1267-06 5f-TAT GGA AAC TTT TCC TCC AAA ATA TCT TCA TTA TGA TGA AGA AAC TTC TCA TCA GCT GCT GTG TGA TAA ATG TCC GCC GGG TAC-3 · (SEQ ID NO ·· 63)

Oligo #1267-07 5-CCG GCG GAC ATT TAT CAC ACA GCA GCT GAT GAG AAG TTT CTT CAT CAT AAT GAA GAT ATT TTG GAG GAA AAG TTT CCA-31 (SEQ ID NO : 64) 經濟部中央標準局貝工消費合作社印裝 (請先閱讀背面之注意事項再填寫本頁) 將pAMG21-huOPG met[22-401]在大腸样菌宿主393中的培養物置於含有20微克/毫升卡那黴素的2XYT培養基中並在謗導前於3〇°C下溫置。在添加合成自動謗導劑Ν·(3-氧基己醯基）-DL-同絲胺酸内酯到培養基中達30毫微克/毫升的最後濃度且將培養物置於30X：或37Χ下培育6小時後即達到從媒體pAMG2l所含luxPR啓動基因謗導出重組基因產物表現之舉。於6小時後，將細菌培養物離心成丸（=30°C 1+6或37°C I+6)。細菌培養物也在謗導之前刻離心成丸（=30°C Prel)或於另一份培養物中不加自動謗導劑而在3〇Ό再培育6小時而得未經謗導（UI)培養物（=30°C UI)。將30°C Prel，30°C UI，30°C 1+6 ’或37°C 1+6諸培養物的細菌丸依B段中所述重新懸浮，溶裂’及以SDS-聚丙烯醯胺凝膠電泳（PAGE)予以分析。將聚丙埽醯胺凝膠用考馬斯藍染色及/或以西方化轉移到硝基纖維素迷用兔子抗-mu OPG-Fc多株抗體依實施例1 0中所述 L ____ -77- 本紙張尺度適用中國國家標準（CNS ) A4規格（2H)x 297公釐） 1221482 A7 B7 五、發明説明（75 ) 進行免疫探測。謗導後的基因產物水平可比擬於未經謗導 (30°C UI)或預謗導（30°C Prel)樣品。 D. t PPG met-r22-4011 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 將編碼鼠（mu)OPG多肽的N -端甲硫胺酸和胺基酸22至401 之DNA序列依下述置於原核生物質體表現媒體pAMG21所含 luxPR啓動基因的控制之下。使用寡核甞酸#1257-16和 #1257_15作爲引子，用質體pRcCMV-Mu OPG DNA作爲模版及以B節中所述熱循環條件實施PCR。將PCR產物依B段中所述予以純化並用Κρη I和B amHI核酸内切限制酶予以劈切。將合成寡核苷酸#126〇_61和#1260-82分別以B段中所述方法磷酸化並使其形成具有Nde I和Κρη I黏狀末端的寡核甞酸聯結子複體。該合成聯結子複體係利用大腸桿菌密碼並提供Ν_端甲硫胺酸。將在寡核苷酸#1260_61與#1260_82之間形成且含有Nde I和Κρη I黏狀末端的經磷酸化聯結子複體及用寡核甞酸引子#1257· 16和# 1257-15產生且經Κρη I和BamHI 消化和純化過的PCR產物用標準方法導引地插置於pAMG21 的Nde I與BamHI邵位之間。將連接混合物以electroporation 用製造商的程序轉形到大腸桿菌宿主393之内。選擇培養出菌株，分離出質體DNA，並進行DNA定序以查證MuOPG met[22_401]基因的DNA序列。使用C段中所述方法測定載有質體pAMG21-MuOPG met[22_4〇l]的393細胞培養物在謗導後的重組muOPG met[22-401]多肽之表現。Oligo # 1267-07 5-CCG GCG GAC ATT TAT CAC ACA GCA GCT GAT GAG AAG TTT CTT CAT CAT AAT GAA GAT ATT TTG GAG GAA AAG TTT CCA-31 (SEQ ID NO: 64) 2XYT medium cooperative printing equipment (read the back surface of the page and then fill Note) the pAMG21-huOPG met [22-401] in E. culture sample was placed in a bacterial host 393 containing 20 g / ml kanamycin And warm at 30 ° C before defamation. The synthetic auto-defense agent N · (3-oxyhexyl) -DL-isoselide was added to the culture medium to a final concentration of 30 ng / ml and the culture was incubated at 30X: or 37X After 6 hours, the performance of deriving the recombinant gene product from the luxPR promoter gene contained in the media pAMG2l was reached. After 6 hours, bacterial cultures were pelleted by centrifugation (= 30 ° C 1 + 6 or 37 ° C I + 6). Engraved pelleted by centrifugation (= 30 ° C Prel) before bacterial culture also abusive or turned to another automatically without culture and incubated slander targeted agent to give 6 hours without slander guide (UI at 3〇Ό ) culture (= 30 ° C UI). Bacteria pellets from 30 ° C Prel, 30 ° C UI, 30 ° C 1 + 6 'or 37 ° C 1 + 6 cultures were resuspended, lysed' and described in SDS-polypropyleneSDS Amine gel electrophoresis (PAGE) was used for analysis. Copolyamide gel stained with Coomassie Blue and / or Westernized to rabbit anti-mu OPG-Fc polyclonal antibody for nitrocellulose fans as described in Example 10 L ____ -77- this paper scale applicable Chinese national standard (CNS) A4 size (2H) x 297 mm) 1221482 A7 B7 V. description of the invention (75) were immunized detection. The level of the defamated gene product can be compared to the undefamated (30 ° C UI) or pre-defamated (30 ° C Prel) sample. D. t PPG met-r22-4011 Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) The N-terminal methionine and amine encoding the mu (OP) peptide the DNA sequence of amino acid 22-401 is placed prokaryotic expression plasmid pAMG21 media contained luxPR promoter by gene under the control of the following. PCR was performed using oligonucleotides # 1257-16 and # 1257_15 as primers, plastid pRcCMV-Mu OPG DNA as a template, and thermal cycling conditions described in Section B. The PCR product was purified as described in paragraph B and cleaved with Kpn I and BamHI endonucleases. The synthetic oligonucleotides # 126〇_61 and # 1260-82 were phosphorylated in the manner described in paragraph B, respectively, and formed into oligonucleotide complexes having sticky ends of Nde I and Kρη I. This synthetic linker complex utilizes the E. coli code and provides N-terminal methionine. Phosphorylated linker complexes formed between oligonucleotides # 1260_61 and # 1260_82 and containing Nde I and Kρη I cohesive ends and generated with oligonucleotide primers # 1257 · 16 and # 1257-15 and The PCR products digested and purified by Kρη I and BamHI were inserted between the Nde I and BamHI sites of pAMG21 in a guided manner using standard methods. The ligation mixture was transformed into E. coli host 393 by electroporation using the manufacturer's procedure. Strains were selected for cultivation, plastid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the MuOPG met [22_401] gene. The performance of the recombinant muOPG met [22-401] polypeptide after defamation was determined using the method described in paragraph C for pAMG21-MuOPG met [22_4l] -containing 393 cell cultures.

Oligo #1257-15 -78-Oligo # 1257-15 -78-

本紙張尺度適用中國國家標準（CNS ) A4規格(210X297公釐T 1221482 A7 B7 五、發明説明（76 ) ··> 5f-TAC GCA CTG GAT CCT TAT AAG CAG CTT ATT TTC ACG GAT TGAAC-3- (SEQ ID NO : 65)This paper size applies to Chinese National Standard (CNS) A4 specifications (210X297 mm T 1221482 A7 B7 V. Description of the invention (76) · > 5f-TAC GCA CTG GAT CCT TAT AAG CAG CTT ATT TTC ACG GAT TGAAC-3- (SEQ ID NO: 65)

Oligo #1257- 1 6 5，-GTG CTC CTG GTA CCT ACC TAA AAC AGC ACT GCA CAG TG-3 丨 (SEQ ID NO · 66)Oligo # 1257- 1 6 5, -GTG CTC CTG GTA CCT ACC TAA AAC AGC ACT GCA CAG TG-3 丨 (SEQ ID NO · 66)

Oligo #1260-61 5f-TAT GGA AAC TCT GCC TCC AAA ATA CCT GCA TTA CGA TCC GGA AAC TGG TCA TCA GCT GCT GTG TGA TAA ATG TGC TCC GGG TAC-3· (SEQ ID NO : 67)Oligo # 1260-61 5f-TAT GGA AAC TCT GCC TCC AAA ATA CCT GCA TTA CGA TCC GGA AAC TGG TCA TCA GCT GCT GTG TGA TAA ATG TGC TCC GGG TAC-3 · (SEQ ID NO: 67)

Oligo #1260-82 5f-CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT CCG GAT CGT AAT GCA GGT ATT TTG GAG GCA GAG TTT CCA-31 (SEQ ID NO : 68) E.鼠 OPG metr32-4011 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 將編碼鼠OPG所含N-端甲硫胺酸和胺基酸3 2至401的DNA 序列依下述置於原核生物質體表現媒體pAMG21中的luxPR 啓動基因之控制下。要完成此舉時，係將合成寡核甘酸 #1267-08和#1267-09以B段中所述方法分別磷酸化並使其形成寡核甞酸聯結子複體。該聯結子複體係利用大腸桿菌密碼且提供N-端甲硫胺酸。將在寡核甞酸#1267-08與#1267-09 之間形成且含有Nde I和Κρη I黏狀末端的經磷酸化聯結子複體及稍早所述經Κρη I和BamHI消化且純化過的PCR產物（參看D段）用標準方法導引地插置於PAMG21的Nde I和BamHI部 -79- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇'〆297公釐) 1221482 A7 B7 五、發明説明（77 ) 位之間。將連接混合物用electroporation以製造商的程序轉形到大腸桿菌宿主393之内。選擇出菌株，分離出質體 DNA，並進行DNA定序以查證muOPG-met[32-401]基因的 DNA序列。使用C段中所述方法測定載有pAMG21重組質體的393細胞培養物在謗導後的重組muOPG-met[32-401]多肽之表現。Oligo # 1260-82 5f-CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT CCG GAT CGT AAT GCA GGT ATT TTG GAG GCA GAG TTT CCA-31 (SEQ ID NO: 68) E. Rat OPG metr32-4011 Economy Printed by the Ministry of Standards and Staff ’s Consumer Cooperative (please read the notes on the back before filling this page). Place the DNA sequence encoding the N-terminal methionine and amino acid 3 2 to 401 in the mouse OPG as follows Under the control of the luxPR promoter in the prokaryotic biomass expression media pAMG21. To do this, the synthetic oligonucleotides # 1267-08 and # 1267-09 were respectively phosphorylated in the manner described in paragraph B and formed into oligonucleotide complexes. This linker complex utilizes the E. coli code and provides N-terminal methionine. Phosphorylated linker complexes formed between oligonucleotides # 1267-08 and # 1267-09 and containing Nde I and κρη I sticky ends and digested and purified with κρη I and BamHI as described earlier The PCR products (see paragraph D) are inserted in the Nde I and BamHI sections of PAMG21 using standard methods. -79- This paper size is in accordance with the Chinese National Standard (CNS) A4 (21〇'297mm) 1221482 A7 B7 V. Description of invention (77). The ligation mixture was transformed into E. coli host 393 using electroporation using the manufacturer's procedure. Strains were selected, plastid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG-met [32-401] gene. The performance of the recombinant muOPG-met [32-401] polypeptide after defamation was measured using the method described in paragraph C for a pAMG21 recombinant plastid-containing 393 cell culture.

Oligo # 1267-08 5f-TAT GGA CCC AGA AAC TGG TCA TCA GCT GCT GTG TGA TAA ATGTGCTCCGGGTAC-3,（SEQ ID NO : 69)Oligo # 1267-08 5f-TAT GGA CCC AGA AAC TGG TCA TCA GCT GCT GTG TGA TAA ATGTGCTCCGGGTAC-3, (SEQ ID NO: 69)

Oligo # 1267-09 5，-CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT GTGGGTCCA-3,（SEQ ID NO : 70) F.鼠 OPG met-lysr22-4011 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 將編碼鼠OPG所含N_端甲硫胺酸接著離胺酸殘基和胺基酸22至401的DNA序列依下述置於原核生物表現媒體 pAMG21中的luxPR啓動基因之控制下。將合成寡核甘酸 #1282-95和#1282_96以B段中所述方法分別磷酸化並使其形成寡核苷酸聯結子複體。該聯結子複體係利用大腸桿菌密碼且提供N-端甲硫胺酸。將在寡核甞酸#1282-95與#1282-96 之間形成且含有Nde I和Κρη I黏狀末端的經磷酸化聯結子複體及D段中所述經Κρη I和BamHI消化且純化過的PCR產物用標準方法導引地插置於pAMG21的Nde I和BamHI部位之間。將連接混合物用electroporation以製造商的程序轉形到大腸 -80- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7__ 五、發明説明（78 ) 桿菌宿主393之内。選擇出菌株，分離出質體DNA，並進行 DNA定序以查證MuOPG_Met-Lys[22-401]基因的DNA序列。使用C段中所述方法測定載有pAMG21重組質體的393細胞培養物在謗導後的重組MuOPG Met-Lys[32-401]多肤之表現。Oligo # 1267-09 5, -CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT GTGGGTCCA-3, (SEQ ID NO: 70) F. Rat OPG met-lysr22-4011 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the precautions on the back before filling this page) Place the DNA sequence encoding the N-terminal methionine in the mouse OPG, followed by the lysine residues and the amines 22 to 401 into the prokaryotes as follows Under the control of the luxPR promoter in the expression media pAMG21. The synthetic oligonucleotides # 1282-95 and # 1282_96 were respectively phosphorylated and formed into oligonucleotide-linked complexes as described in paragraph B. This linker complex utilizes the E. coli code and provides N-terminal methionine. Phosphorylated linker complexes formed between oligonucleotides # 1282-95 and # 1282-96 and containing Nde I and κρη I cohesive ends, and described with κρη I and BamHI digestion and purification Passed PCR products were inserted between the Nde I and BamHI sites of pAMG21 in a guided manner using standard methods. The connection mixture is transformed to the large intestine by electroporation according to the manufacturer's procedure.- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 A7 B7__ 5. Description of the invention (78) within the Bacillus host 393 . A strain was selected, plastid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the MuOPG_Met-Lys [22-401] gene. The performance of the recombinant MuOPG Met-Lys [32-401] polypeptide after defamation was measured using the method described in paragraph C for 393 cell cultures carrying pAMG21 recombinant plastids.

Oligo # 1282-95 5f-TAT GAA AGA AAC TCT GCC TCC AAA ATA CCT GCA TTA CGA TCC GGA AAC TGG TCA TCA GCT GCT GTG TGA TAA ATG TGC TCC GGGTAC-3- (SEQ ID NO : 71)Oligo # 1282-95 5f-TAT GAA AGA AAC TCT GCC TCC AAA ATA CCT GCA TTA CGA TCC GGA AAC TGG TCA TCA GCT GCT GTG TGA TAA ATG TGC TCC GGGTAC-3- (SEQ ID NO: 71)

Oligo # 1282-96 5'-CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT CCG GAT CGT AAT GCA GGT ATT TTG GAG GCA GAG TTT CTT TCA-3' (SEQ ID NO : 72) G.鼠 OPG met_lys-niisW22_4011 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 將編碼鼠 OPG 的 N-端殘基 Met-Lys_His-His_His-His-His-His-His(=MKH)接著胺基酸2 2到401之DNA序列依下述置於原核生物表現媒體pAMG21所含luxPR啓動基的控制之下。使用寡核苷酸#1300-50和#1257-15作爲引子及質體pAMG21-muOPG_met[22_401] DNA作爲模版進行PCR。熱循環條件爲 B段中所述者。將所得PCR產物剪開，純化，用Nde I和 BamHI核酸内切限制酶切劈並純化。將用寡核甞酸引子 #1300-50和#1257-15產生且經Nde I和BamHI消化和純化過的 PCR產物用標準DNA方法導引地插置於pAMG21的Nde I和 BamHI部位之間。將連接混合物以electroporation用製造商 -81 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（79 ) 的程序轉形到大腸桿菌宿主393之内。選擇培養出菌株，分離出質體DNA，並進行DNA定序以查證muOPG-MKH[22-401] 基因的DNA序列。使用C段中所述方法測定載有重組p AMG21質體的經轉形 393培養物在謗導後的重組MuOPG-MKH[22-401]多肽之表現0Oligo # 1282-96 5'-CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT CCG GAT CGT AAT GCA GGT ATT TTG GAG GCA GAG TTT CTT TCA-3 '(SEQ ID NO: 72) G. Rat OPG met_lys -niisW22_4011 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling this page) The N-terminal residue Met-Lys_His-His_His-His-His-His-His (= MKH) The DNA sequences of amino acids 22 to 401 are placed under the control of the luxPR promoter contained in the prokaryotic expression media pAMG21 as follows. PCR was performed using oligonucleotides # 1300-50 and # 1257-15 as primers and plastid pAMG21-muOPG_met [22_401] DNA as a template. Thermal cycling conditions are those described in paragraph B. The resulting PCR product was cut open, purified, cleaved with Nde I and BamHI endonuclease restriction enzymes, and purified. PCR products produced with oligonucleotide primers # 1300-50 and # 1257-15 and digested and purified with Nde I and BamHI were inserted between the Nde I and BamHI sites of pAMG21 using standard DNA methods. The connection mixture is electroporation manufacturer -81-This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 V. The procedure of invention description (79) is transformed into E. coli host 393. The strain was selected and cultured, plastid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG-MKH [22-401] gene. The expression of recombinant MuOPG-MKH [22-401] peptides after transformation in 393 cultures containing recombinant p AMG21 plastids was determined using the method described in paragraph C. 0

Oligo # 1300-50 5f-GTT CTC CTC ΑΤΑ TGA AAC ATC ATC ACC ATC ACC ATC ATG AAA CTC TGC CTC CAA AAT ACC TGC ATT ACG AT-3* (SEQ ID NO : 73)Oligo # 1300-50 5f-GTT CTC CTC ΑΤΑ TGA AAC ATC ATC ACC ATC ACC ATC ATG AAA CTC TGC CTC CAA AAT ACC TGC ATT ACG AT-3 * (SEQ ID NO: 73)

Oligo # 1257-15 (參看D段） Η·鼠 OPG met_lvsr22-4011 (his)? 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 將編碼鼠OPG所含N-端met-lys，胺基酸22至401，及胺基酸401後面所接七個組胺酸殘基的DNA序列（=muOPG MK [22-401]·Η7)依下文所述置於原核生物表現媒體pAMG21所含 lux PR啓動基因之控制下。使用寡核苷酸#1300_49和#1300-51作爲引子及用pAMG21-muOPG met[22_401] DNA作爲模版進行PCR。其熱循環條件爲B段中所述者。將所.得PCR樣品置於瓊脂凝膠上分離，將PCR產物剪開，純化，用Nde I和 BamHI核酸内切限制酶切劈並純化。經Nde I和BamHI消化和純化過的PCR產物使用標準DNA方法導引地插置於pAMG21 的Nde I和BamHI部位之間。將連接混合物以electroporation -82 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 _ B7__ 五、發明説明（80 ) 用製造商的程序轉形到大腸样菌宿主393之内。選擇培養出菌株，分離出質體DNA，並進行DNA定序以查證muOPG-ΜΚΗ[22·401]·Η7基因的 DNA序列。使用C段中所述方法測定載有重組pAMG21質體的經轉形 393培養物在謗導後的重組muOPG-MKH[22_401]-H7多肽之表現。Oligo # 1257-15 (see paragraph D) 鼠 · Mouse OPG met_lvsr22-4011 (his)? Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). The code included in the mouse OPG The DNA sequences of the N-terminal met-lys, amino acids 22 to 401, and the seven histidine residues following amino acid 401 (= muOPG MK [22-401] · Η7) are placed as described below Under the control of the lux PR promoter gene contained in the prokaryotic expression media pAMG21. PCR was performed using oligonucleotides # 1300_49 and # 1300-51 as primers and pAMG21-muOPG met [22_401] DNA as a template. Its thermal cycling conditions are those described in paragraph B. The obtained PCR samples were separated on an agar gel, the PCR products were cut apart, purified, cleaved with Nde I and BamHI endonucleases, and purified. The Nde I and BamHI digested and purified PCR products were guidedly inserted between the Nde I and BamHI sites of pAMG21 using standard DNA methods. The connection mixture is electroporation -82-this paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 _ B7__ V. Description of the invention (80) Use the manufacturer's procedure to transform to the coliform host 393 Inside. The strain was selected and cultured, plastid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG-MKΗ [22 · 401] · Η7 gene. The performance of the transformed muOPG-MKH [22_401] -H7 polypeptide after transformation with the transformed 393 culture containing the recombinant pAMG21 plastid was determined using the method described in paragraph C.

Oligo # 1300-49 5'-CTT CTC CTC ΑΤΑ TGA AAG AAA CTC TGC CTC CAA AAT ACC TGC A-3f (SEQ ID NO ： 74)Oligo # 1300-49 5'-CTT CTC CTC ΑΑΑ TGA AAG AAA CTC TGC CTC CAA AAT ACC TGC A-3f (SEQ ID NO: 74)

Oligo # 1300-51 5f-TAC GCA CTG GAT CCT TAA TGA TGG TGA TGG TGA TGA TGT AAG CAG CTT ATT TTC ACG GAT TGA ACC TGA TTC CCT Α·3· (SEQ ID NO : 75) I.鼠 OPG meU27-4〇n 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 將編碼鼠OPG所含N-端甲硫胺酸和胺基酸27至401的DNA 序列依下述置於原核生物表現媒體pAMG21中的lux PR啓動基因之控制下。用寡核酸苷酸#1309-74和#1257-15作爲引子及用質體pAMG21-muOPG met[22-401] DNA作爲模版。熱循環條件爲B段中所述者。將所得PCR樣品置於瓊脂凝膠上分離，將PCR產物剪開，純化，用Nde I和BamHI核酸内切限制酶切劈並純化。將經Nde I和BamHI消化和純化過的PCR產物用標準方法導引地插置於pAMG21的Nde I與BamHI部位之間。將連接混合物以electroporation用製造商的程序轉形到 -83- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）經濟部中央標準局員工消費合作社印裂 1221482 A7 B7 五、發明説明（81) 大腸样菌宿主393之内。選擇培養出菌株，分離出質體 DNA，並進行DNA定序以查證muOPG-met[27-401]基因的 DNA序列。使用C段中所述方法測定載有重組p AMG21質體的經轉染 393培養物於謗導後的重組muOPG-met[22-401]多肽之表現。 Oligo # 1309-74 5LGTT CTC CTC ΑΤΑ TGA AAT ACC TGG ATT ACG ATC CGG AAA CTGGTCAT-3,（SEQ ID NO : 76)Oligo # 1300-51 5f-TAC GCA CTG GAT CCT TAA TGA TGG TGA TGG TGA TGA TGT AAG CAG CTT ATT TTC ACG GAT TGA ACC TGA TTC CCT Α · 3 · (SEQ ID NO: 75) I. Rat OPG meU27-4 〇n Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) The DNA sequence encoding the N-terminal methionine and amino acids 27 to 401 contained in the mouse OPG The lux PR promoter gene placed in the prokaryotic expression media pAMG21 is described under control. Oligonucleotides # 1309-74 and # 1257-15 were used as primers, and pAMG21-muOPG met [22-401] DNA was used as a template. Thermal cycling conditions are those described in paragraph B. The obtained PCR samples were separated on an agar gel, and the PCR products were cut apart, purified, cleaved with Nde I and BamHI endonucleases, and purified. The PCR products digested and purified by Nde I and BamHI were inserted between the Nde I and BamHI sites of pAMG21 in a guided manner using standard methods. The connection mixture is transformed to -83 by the manufacturer's procedure for electroporation. This paper size is applicable to the Chinese National Standard (CNS) A4 (210X297 mm). Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. (81) Within coliform host 393. The strain was selected and cultured, plastid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG-met [27-401] gene. The performance of the recombinant muOPG-met [22-401] polypeptide after transfection with the transfected 393 culture containing the recombinant p AMG21 plastid was determined using the method described in paragraph C. Oligo # 1309-74 5LGTT CTC CTC ΑΑΑ TGA AAT ACC TGG ATT ACG ATC CGG AAA CTGGTCAT-3, (SEQ ID NO: 76)

Oligo # 1257-15 (參看D段） J·人類 OPG met『27-4011 將編碼人類OPG所含N-端甲硫胺酸和胺基酸27至401的 DNA序列依下述置於原核生物表現媒體pAMG21中的lux PR 啓動基因之控制下。使用寡核酸苷酸#1309-75和#1309-76作爲引子及用質體pAMG21-muOPG-met[22-401] DNA作爲模版進行PCR。熱循環條件爲B段中所説明者。將所得PCR樣品置於瓊脂糖凝膠上分離，將PCR產物剪下，純化，用Asel和 BamHI核酸内切限制酶予以限制消化，並純化。將上述經 Asel和BamHI消化且純化過的PCR產物用標準方法導引地插置於pAMG21的Nde I和BamHI部位之間。將連接混合物以 electroporation用製造商的程序轉形到大腸桿菌宿主393之内。選擇培養出菌株，分離出質體DNA，並進行DNA定序以查證huOPG-met[27-401]基因的DNA序列。 -84- 本紙張尺度適用中國國家標準（CNS ) A4規格（2丨〇'〆297公釐） (請先閱讀背面之注意事項再填寫本頁) •裝· 48 1X 2 12 A7 _B7__ 五、發明説明（S2 ) 使用C段中所述方法測定載有重組pAMG21質體的經轉染 393細胞在謗導後的重組huOPG-met[27-401]多肽之表現。 (請先閲讀背面之注意事項再填寫本頁)Oligo # 1257-15 (see paragraph D) J. Human OPG met "27-4011 The DNA sequence encoding the N-terminal methionine and amino acids 27 to 401 contained in human OPG was placed in a prokaryotic expression as follows Under the control of the lux PR promoter in the media pAMG21. PCR was performed using oligonucleotides # 1309-75 and # 1309-76 as primers and pAMG21-muOPG-met [22-401] DNA as a template. Thermal cycling conditions are those described in paragraph B. The obtained PCR sample was separated on an agarose gel, the PCR product was cut out, purified, digested with Asel and BamHI endonucleases, and purified. The Asel and BamHI digested and purified PCR products described above were inserted between the Nde I and BamHI sites of pAMG21 in a guided manner using standard methods. The ligation mixture was transformed into E. coli host 393 using manufacturer's procedures using electroporation. The strain was selected and cultured, plastid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the huOPG-met [27-401] gene. -84- This paper size applies to Chinese National Standard (CNS) A4 specification (2 丨〇'〆297mm) (Please read the precautions on the back before filling this page) • Packing · 48 1X 2 12 A7 _B7__ V. Invention Explanation (S2) The method described in paragraph C was used to determine the expression of recombinant huOPG-met [27-401] polypeptide in transfected 393 cells carrying recombinant pAMG21 plastids. (Please read the notes on the back before filling this page)

Oligo # 1309-75 5，-GTT CTC CTA TTA ATG AAA TAT CTT CAT TAT GAT GAA GAA ΑΟΓΊΆ (SEQ ID NO : 77)Oligo # 1309-75 5, -GTT CTC CTA TTA ATG AAA TAT CTT CAT TAT GAT GAA GAA ΑΟΓΊΆ (SEQ ID NO: 77)

Oligo #1309-76 5f-TAC GCA CTG GAT CCT TAT AAG CAG CTT ATT TTT ACT GAT T_3,（SEQ ID NO : 78) K.鼠 OPG met『22-18〇l 經濟部中央標準局員工消費合作社印製將編碼鼠OPG所含N-端甲硫胺酸和胺基酸22至180的DNA 序列依下述置於原核生物質體表現媒體pAMG21中的lux PR 啓動基因之控制下。使用寡核酸甞酸#1309-72和#1309_73作爲引子及用質體pAMG21-muOPG met[22-401] DNA作爲模版進行PCR。熱循環條件爲B段中所述者。將所得PCR樣品置於瓊脂糖凝膠上分離，將PCR產物剪下，純化，用Nde I和 BamHI核酸内切限制酶予以限制消化。將上述經Nde I和 BamHI消化且純化過的PCR產物用標準方法導引地插置於 pAMG21的Nde I與BamHI部位之間。將連接混合物用 electroporation以製造商的程序轉形到大腸样菌宿主393之内。選擇出菌株，分離出質體DNA，並進行DNA定序以查證muOPG-met[22-180]基因的 DNA序列。使用C段中所述方法測定載有重組P AMG21質體的經轉形 393培養物於謗導後的重組muOPG_met[22-180]多肽之表 -85 - 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X297公釐） 1221482 A7 B7 五、發明説明（83 ) 現0Oligo # 1309-76 5f-TAC GCA CTG GAT CCT TAT AAG CAG CTT ATT TTT ACT GAT T_3, (SEQ ID NO: 78) K. Rat OPG met 『22-18〇l Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs The DNA sequences encoding the N-terminal methionine and amino acids 22 to 180 contained in the murine OPG were placed under the control of the lux PR promoter gene in the prokaryotic biomass expression media pAMG21 as described below. PCR was performed using oligonucleotides # 1309-72 and # 1309_73 as primers and pAMG21-muOPG met [22-401] DNA as a template. Thermal cycling conditions are those described in paragraph B. The obtained PCR samples were separated on an agarose gel, the PCR products were cut out, purified, and restricted digestion with Nde I and BamHI endonucleases. The PCR products digested and purified by Nde I and BamHI as described above were inserted between the Nde I and BamHI sites of pAMG21 in a guided manner using standard methods. The ligation mixture was transformed into the coliform host 393 using electroporation according to the manufacturer's procedure. A strain was selected, plastid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG-met [22-180] gene. Use the method described in paragraph C to determine the recombined muOPG_met [22-180] peptide of the transformed 393 culture containing the recombinant P AMG21 plastid. Table -85-This paper applies Chinese national standards (CNS) A4 specification (21 × 297 mm) 1221482 A7 B7 V. Description of the invention (83) Now 0

Oligo # 1309-72 5f-GTT CTC CTC ΑΤΑ TGG AAA CTC TGC CTC CAA AAT ACC TGC A-3· (SEQ ID NO : 79)Oligo # 1309-72 5f-GTT CTC CTC ΑΑΑ TGG AAA CTC TGC CTC CAA AAT ACC TGC A-3 · (SEQ ID NO: 79)

Oligo #1309-73 5f-TAC GCA CTG GAT CCT TAT GTT GCA TTT CCT TTC TGA ATT AGCA-31 (SEQ ID NO ： 80) L·鼠 OPG met『27-18(n 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 將編碼鼠OPG的N-端甲硫胺酸和胺基酸27至180之DNA序列依下文所述置於原核生物表現媒體pAMG21中的lux PR啓動基因的控制之下。使用寡核酸苷酸#1309-74(參看I節）和 #1309-73(參看K節）作爲引子及用質體pAMG21-muOPG met [22-401] DNA作爲模版進行PCR。熱循環條件爲B節中所述者。將所得PCR樣品置於瓊脂糖凝膠上分離，將PCR產物剪下，純化，用Nde I和BamHI核酸内切限制酶予以限制消化，並純化。將上述經Nde I和BamHI消化且純化過的PCR產物用標準方法導引地插置在pAMG21的Nde I和BamHI部位之間。將連接混合物用electroporation以製造商的程序轉形到大腸桿菌宿主393之内。選擇出菌株，分離出質體DNA，並進行DNA定序以查證muOPG-met[27-180]基因的DNA序列。使用C段中所述方法測定載有重組p AMG21質體的經轉形 393細胞培養物在謗導後的重組mu〇PG met[27-180]多肽之表現0 -86- 本紙張尺度適用中國國家標準（CNS ) A4規格（2丨〇'〆297公釐）經濟部中央標準局員工消費合作社印製 1221482 A7 _B7__ 五、發明説明（84 ) M·氣〇PG met『22-1891 和met『22-1941 將編碼鼠OPG所含N-端甲硫胺酸和胺基酸22至189，或22 到194的DNA序列依下述置於原核生物表現媒體pAMG21所含 lux PR啓動基因之控制下，使用B節中所述方法將合成寡核甞酸配對 #1337-92 和 #1337-93(=muOPG-189聯結子）或 #1333· 57和#1333-58(=muOPG-194聯結子）分別磷酸化並使其形成寡核甞酸聯結子複體配對。將經純化的質體PAMG21-muOPG met[22-401] DNA用ΚρηΙ和BspEI核酸内切限制酶予以切劈並將所得DNA片段置於瓊脂糖凝膠上分離。用標準重組DNA方法分離出〜413 bp B片段。將在寡核核苷酸 #1337-92與#13 37_93(muOPG-l89 聯結子）或寡核苷酸 #1333-57和#1333-58(muOPG-194聯結子）之間形成且含有BspEI和 BamHI黏性末端之經磷酸化寡核甞酸聯結子複體，及上述分離出且經ΚρηΙ和BspEI核酸内切限制酶消化過的質體 pAMG21-muOPG-met [22-401]所含〜413 bp B片段使用標準方法導引地插置在pAMG21-muOPG_met [22-401]的ΚρηΙ與 BamHI部位之間。將各連接混合物以electroporation用製造商的程序轉形到大腸桿菌宿主393之内。選擇培養出株系，分離出質體DNA，並進行DNA定序以查證muOPG_met [22-189]或 muOPG-met [22-194]基因的 DNA序列。用C節中所述方法測定轉形到393細胞内的重組pAMG21質體之重組muOPG-met[22-189]和 muOPG-met[22-194]多肽之表現0Oligo # 1309-73 5f-TAC GCA CTG GAT CCT TAT GTT GCA TTT CCT TTC TGA ATT AGCA-31 (SEQ ID NO: 80) L. Rat OPG met 『27-18 (n Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Ltd. (Note read back surface of the page and then fill) encoding the murine OPG N- terminal methionine and amino acids sequences of DNA 27 to 180. prokaryotes by expression disposed below the medium in pAMG21 lux gene under the control of PR promoter. guanylate using oligonucleotides # 1309-74 (see section I) and # 1309-73 (see section K) as primers and met [22-401] using plasmid pAMG21-muOPG DNA as template for PCR. thermal cycling conditions were those described in section B. the resulting PCR sample was placed agarose gel, the PCR product was cut out, purified, cut with Nde I restriction enzyme BamHI nucleic acid and be limited within digested purification. the above-described via Nde I and BamHI digested and purified PCR product using standard techniques guide interposed between the Nde I and BamHI sites of pAMG21. the ligation mixture was used to electroporation of the program manufacturer Transformation into E. coli 393 within the host. selected strain, the plasmid DNA isolated, and DNA Sequencing to verify that muOPG-met [27-180] DNA sequence of the gene. The method used in section C measured by carrying Transformation of recombinant plasmid p AMG21 393 recombinant cell culture mu〇PG met in the guide slander [27-180] Polypeptide performance 0 -86- This paper size is applicable to Chinese National Standard (CNS) A4 specification (2 丨〇297mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 1221482 A7 _B7__ V. Invention Explanation (84) M · qi〇PG met "22-1891 and met" 22-1941 will encode the N-terminal methionine and amino acids 22 to 189, or 22 to 194 of the mouse OPG prokaryotic expression of said medium disposed at pAMG21 lux PR promoter contained in the control gene, using the methods set forth in section B synthetic oligonucleotide pair # 1337-92 Chang acid and # 1337-93 (= muOPG-189 linker), or * 57 # 1333 and # 1333-58 (= muOPG-194 linker) were phosphorylated and allowed to form an acid linker oligonucleotide duplexes Chang pairing. the met [22-401] DNA purified plasmid pAMG21-muOPG ΚρηΙ with a nucleic acid and BspEI endonuclease restriction enzymes and the resulting DNA to be chop agarose gel fragment is placed. A ~ 413 bp B fragment was isolated using standard recombinant DNA methods. The formation of the oligonucleotide or polynucleotide # 1337-92 and # 13 37_93 (muOPG-l89 linker) oligonucleotides # 1333-57 and # 1333-58 between (muOPG-194 linker) containing BspEI and and phosphorylated oligonucleotide Chang acid linker duplexes cohesive ends of BamHI, and the separation and through the inner ΚρηΙ and BspEI restriction endonucleases digested plasmid pAMG21-muOPG-met [22-401] contained ~413 bp B fragment using standard methods interposed between the guide pAMG21-muOPG_met [22-401] ΚρηΙ and the BamHI site. Each ligation mixture was used to electroporation of the program manufacturer Transformation into the E. coli host 393. Strains were selected for cultivation, plastid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG_met [22-189] or muOPG-met [22-194] gene. The expression of recombinant muOPG-met [22-189] and muOPG-met [22-194] peptides of recombinant pAMG21 plastid transformed into 393 cells was determined by the method described in Section C. 0

Oligo # 1337-92 -87 - 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇'〆297公釐） (請先聞讀背面之注意事項再填寫本頁)Oligo # 1337-92 -87-This paper size applies to Chinese National Standard (CNS) A4 (21〇'〆297mm) (Please read the precautions on the back before filling out this page)

1221482 A7 B7 五、發明説明（85 ) S'-CCGGAAACAGAT ATTGAG-31 (SEQ ID NO ： 81)1221482 A7 B7 V. Description of the invention (85) S'-CCGGAAACAGAT ATTGAG-31 (SEQ ID NO: 81)

Oligo # 1337-93 5，-GAT CCT CAT TAT CTG TTT-3· (SEQ ID NO : 82)Oligo # 1337-93 5, -GAT CCT CAT TAT CTG TTT-3 · (SEQ ID NO: 82)

Oligo # 1333-57 5f-CCG GAA ACA GAG AAG CCA CGC AAA AGT AAG-3f (SEQ ID NO ： 83)Oligo # 1333-57 5f-CCG GAA ACA GAG AAG CCA CGC AAA AGT AAG-3f (SEQ ID NO: 83)

Oligo # 1333-58 5.-GAT CCT TAC TTT TGC GTG GCT TCT CTG TTT-3,（SEQ ID NO ： 84) N.鼠 OPG met『27-1891 和met [27-1941 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 將編碼鼠OPG所含N-端甲硫胺酸和胺基酸22至189，或27 至194的DNA序列依下文所述置於原核生物表現媒體pAMG21 所含lux PR啓動基因的控制之下。將含有BspEI和BamHI黏性末端經磷酸化的寡核苷酸聯結子，nmuOPG-189聯結子"或 "muOPG-194聯結子”（參看Μ節），及分離出的經ΚρηΙ和 BspHI核酸内切限制酶消化過的質體pAMG21-muOPG_met [22-401]所含〜413 bp B片段用標準方法導引地插置於質體 pAMG21-muOPG-met [27-401]的ΚρηΙ和 BamHI部位之間。將每一連接混合物以electroporation用製造商的程序轉形到大腸桿菌宿主393之内。選擇出菌株，分離出質體DNA，並進行 DNA 定序以查證 muOPG met [27-189]或 muOPG met [27-194]基因之DNA序列。用C節中所述方法測定載著重組pAMG21質體的393細胞經 -88- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7__ 五、發明説明（86 ) 謗導後的重組muOPG met[27-189]或muOPG met[27-194]之表現。 Ο.人類 OPG met『22-1851，met Γ22-1891，metr22-1941 經濟部中央標準局員工消費合作衽印製 (請先閲讀背面之注意事項再填寫本頁) 將編碼人類OPG多肽的N-端甲硫胺酸及胺基酸22至185， 22至189，或22到194之DNA序列依下述置於原核生物表現媒體pAMG21所含lux PR啓動基因的控制之下，將合成寡核甞酸配對 #1331-87 和 #1331-88(=huOPG_185聯結子），#133 卜 89 和 #1331-90(=huOPG_189 聯結子），或 #1331-91 & #1331-92 (=huOPG-194聯結子）分別磷酸化且使用B節所述方法予以分別形寡核苷酸聯結子複體。將經純化的pAMG21-huOPG-met[27-401]質體DNA用ΚρηΙ和Nael核酸内切限制酶予以限制消化並將所得DNA片段置於瓊脂糖凝膠上分離。用標準重組DNA方法分離出〜407 bp B片段。將在寡核核苷酸 #1337-87 與 #1331-88(huOPG_185 聯結子），寡核苷酸 #1331-89 與#1331_90(huOPG-189聯結子），或寡核甞酸#1331-91與 #133192(huOPG194聯結子）[每一聯結子含有NdeI和BamHI 黏性末端]之間形成的經磷酸化寡核苷酸聯結子複體，及上述分離出的經ΚρηΙ和Ndel核酸内切限制酶消化過之質體 pAMG21_huOPG-met [27-401]所含〜407 bp B片段經由使用標準方法導引地插置在質體pAMG21-huOPG-met[22-401]的 ΚρηΙ和BamHI部位之間。將每一連接物以electroporation用製造商的程序轉形到大腸桿菌宿主393内。選擇出菌株，分離出質體DNA，並進行DNA定序以查證1111〇?0-1^1[22-185]，huOPG-met [22-189]，或 huOPG-met [22-194]基因的 -89 - 本紙ίλ尺度適用中國國家標準（CNS ) A4規格（210X297公釐了 1221482 A7 B7 五、發明説明（87 ) DNA序列。 (請先閱讀背面之注意事項再填寫本頁) 用C節所述方法測定載有重組pAMG21質體的經轉形393細胞在謗導後的重組 huOPG-met[22-185]，huOPG-met[22-189] 或 huOPG_met[22-194]之表現。Oligo # 1333-58 5.-GAT CCT TAC TTT TGC GTG GCT TCT CTG TTT-3, (SEQ ID NO: 84) N. Rat OPG met "27-1891 and met [27-1941 Staff Consumption of Central Standards Bureau, Ministry of Economic Affairs Printed by the cooperative (please read the notes on the back before filling out this page) Place the DNA sequence encoding the N-terminal methionine and amino acids 22 to 189, or 27 to 194 of the mouse OPG as described below The prokaryotic expression media pAMG21 is under the control of the lux PR promoter. A phosphorylated oligonucleotide linker containing the BspEI and BamHI cohesive ends, the nmuOPG-189 linker " or " muOPG-194 linker " The ~ 413 bp B fragment contained in the endoplasmic digested pAMG21-muOPG_met [22-401] was inserted in the κρηΙ and BamHI sites of pAMG21-muOPG-met [27-401] in a guided manner using standard methods. Between. Each ligation mixture was transformed into E. coli host 393 using the manufacturer's procedure using electroporation. A strain was selected, plastid DNA was isolated, and DNA sequencing was performed to verify muOPG met [27-189] or DNA sequence of the muOPG met [27-194] gene. 393 cells carrying recombinant pAMG21 plastids were determined by the method described in Section C. -88- This paper is in accordance with China National Standard (CNS) A4 (210X 297 mm) ) 1221482 A7 B7__ V. Description of the invention (86) The performance of the reorganized muOPG met [27-189] or muOPG met [27-194] after defamation. -1941 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the back first) Note: Please fill in this page again.) Place the N-terminal methionine and amino acids 22 to 185, 22 to 189, or 22 to 194 encoding human OPG polypeptide in the prokaryotic expression media pAMG21 as follows. Under the control of the lux PR promoter gene, pair synthetic oligonucleotides # 1331-87 and # 1331-88 (= huOPG_185 linker), # 133 bu89 and # 1331-90 (= huOPG_189 linker), or # 1331-91 &# 1331-92 (= huOPG-194 linker) were separately phosphorylated and the oligonucleotide linker complex was shaped separately using the method described in Section B. Purified pAMG21-huOPG-met [ 27-401] Plastid DNA was digested with κρηΙ and Nael endonuclease restriction enzymes and the resulting DNA fragments were separated on an agarose gel. ~ 407 bp B fragments were isolated using standard recombinant DNA methods. Nucleotides # 1337-87 and # 1331-88 (huOPG_185 linker), oligonucleotides # 1331-89 and # 1331_90 (huOPG-189 linker), or oligonucleotides # 1331-91 and # 133192 ( huOPG194 linker) [each linker contains NdeI and BamHI sticky ends] phosphorylated oligonucleotide linker complex formed between the above-mentioned components The ~ 407 bp B fragment contained in the plastid pAMG21_huOPG-met [27-401] digested with κρηΙ and Ndel endonuclease restriction enzymes was inserted in plastid pAMG21-huOPG-met [22] using a standard method. -401] between the κρηΙ and BamHI sites. Each linker was transformed into E. coli host 393 using the manufacturer's procedure using electroporation. Select the strain, isolate the plastid DNA, and perform DNA sequencing to verify the 1111? 0-1 ^ 1 [22-185], huOPG-met [22-189], or huOPG-met [22-194] gene的 -89-This paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm 1221482 A7 B7) 5. Description of the invention (87) DNA sequence. (Please read the precautions on the back before filling this page) Use section C The method measures the performance of recombinant huOPG-met [22-185], huOPG-met [22-189], or huOPG_met [22-194] after transformation of transformed 393 cells carrying recombinant pAMG21 plastids.

Oligo # 1331-87 5，-TAT GTT AAT GAG -3’（SEQ ID NO : 85)Oligo # 1331-87 5, -TAT GTT AAT GAG -3 ’(SEQ ID NO: 85)

Oligo # 1331-88 5.-GAT CCT CAT TAA CA-3,（SEQ ID NO : 86)Oligo # 1331-88 5.-GAT CCT CAT TAA CA-3, (SEQ ID NO: 86)

Oligo # 1331-89 5，-TAT GTT CCG GAA ACA GTT AAG-31 (SEQ ID NO : 87) Oligo # 1331-90 5，-GAT CCT TAA CTG TTT CCG GAA CA-3* (SEQ ID NO : 88)Oligo # 1331-89 5, -TAT GTT CCG GAA ACA GTT AAG-31 (SEQ ID NO: 87) Oligo # 1331-90 5, -GAT CCT TAA CTG TTT CCG GAA CA-3 * (SEQ ID NO: 88)

Oligo # 1331-91 5丨-TAT GTT CCG GAA ACA GTG AAT CAA CTC AAA AAT AAG-31 (SEQ ID NO ·· 89)Oligo # 1331-91 5 丨 -TAT GTT CCG GAA ACA GTG AAT CAA CTC AAA AAT AAG-31 (SEQ ID NO · 89)

Oligo # 1331-92 經濟部中央標準局員工消費合作社印製 5，-GAT CCT TAT TTT TGA GTT GAT TCA CTG TTT CCG GAA GA_3,（SEQ ID NO : 90) P.人類 OPG met『27-1851，met『27-1891，met『27-1941 將編碼人類OPG多肽所含的N·端甲硫胺酸和胺基酸27至 185，27至189，或27到194的DNA序列依下文所述置於原核生 •90- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇'〆297公釐） 1221482 A7 B7 五、發明説明（88 ) 物表現媒體pAMG21所含lux PR啓動基因的控制之下。將各含Nde I和BamHI黏狀末端的經磷酸化寡核甞酸聯結子 ”huOPG_185聯結子’’，”huOPG-189聯結子"或"huOPG-194聯結子"（參看0節），及分解出的經KpnI和NdeI核酸内切酶消化過之質體pAMG21-huOPG-met [27-401]所含〜407 bp B片段 (參看0節），使用標準方法導引地插置於質體PAMG21· huOPG-met [27-401]的ΚρηΙ和BamHI部位之間（參看J節）。將各連接物用electroporation以製造商的程序轉形到大腸桿菌宿主393之内。選擇出菌株，分離出質體DNA，並進行DNA 定序以查證huOPG-met [27-185]，huOPG-met [27-189]，或 huOI>G-met[27-194]基因的DNA序列。使用C節中所述方法測定轉形到393細胞内的重組pAMG21 質體之重組 huOPG-met[27-185]，huOPG-met[27_189]，和 huOPG-met[27-194]的表現。 Q·鼠OPG metr27-401UP33E，G36S，A45P) 經濟部中央標準局員Η消費合作衽印製 (請先閱讀背面之注意事項再填寫本頁) 將編碼人類OPG所含N-端甲硫胺酸和胺基酸27至48及鼠 OPG所含胺基酸殘基4 9至401的DNA序列依下述置於原核生物表現媒體pAMG21所含lux PR啓動基因的控制之下。將純化出的質體 pAMG21-huOPG-met[27-401] DNA(參看 J 節）用 Aatll和ΚρηΙ核酸内切限制酶切劈並用標準重組DNA方法從瓊脂糖凝膠分離出〜1075 bp Β片段。此外，將質體pAMG21-muOPG-met[22-401] DNA(參看 D 節）用 ΚρηΙ和BamHI核酸内切限制酶消化並依上述分離出〜1064 bp B片段。用標準重組 DNA方法將分離出且含有Aatll & ΚρηΙ黏性末端的〜1〇75 bp -91 - 本紙張尺度適用中國國家標準（ CNS ) A4規格（210X297公釐） 2 48 1X 2 12 A7 B7_ 五、發明説明（89) pAMG21-huOPG-met[27-401] P艮制片段（參看上文），含有 ΚρηΙ和BamHI黏性末端的〜1064 bp pAMG21-muOPG-met[22-401]限制片段及含有Aatll和BamHI黏性末端且對應於 pAMG21在Aatll和BamHI之間的核酸序列之〜5043 bp限制片段予以連接起來。將連接體經由electroporatino用製造商的程序轉形到大腸桿菌宿主393内。選擇出菌株，並用標準 DNA方法查證質體中的重組***體之存在。muOPG-27_401 (P33E，G36S，A45P)基因。在muOPG中脯胺酸-33變成穀胺酸-33，甘胺酸-36變成絲胺酸-36，及丙胺酸-45變成脯胺酸-45諸胺基酸改變係由muOPG殘基2 7至4 8被huOPG殘基2 7至 48取代所促成的。用C節中所述方法測定載有重組pAMG21質體的經轉形393] 細胞之重組muOPG_met[27-401] (P33E ’ G36S ’ A45P)之表現。 R.鼠OPG met-lvs-(his)7-ala-ser-(asp)i-lys[22-401](A45T) 經濟部中央榡準局員工消費合作衽印製 (請先閱讀背面之注意事項再填寫本頁) 將編碼鼠OPG多肽所含N-端His標記和腸激酶辨識序列（自 NH2 端至 COOH 端）：Met-Lys-His-His-His-His-His-His-His-Ala_Ser-Asp_Asp-Asp-Asp-Lys(=HEK) ’ 接著胺基酸22 至 401 的DNA序列依下述置於被lat限遏物調節的Ps4啓動基因之控制下。將pAMG22-His(參看A節）用Nhe I和BamHI核酸内切限制酶予以消化並用標準重組DNA方法從瓊脂糖凝膠分離出大片段（A片段）。將寡核甞酸#1282-91和#1282-92用前面所述方法予以分別磷酸化並使其形成寡核苷酸聯結子複體 (參看B節）。使用標準重組DNA方法將在寡核甞酸#1282-91 與#1282-92之間形成且含有Nhe I和ΚρηΙ黏狀末端的經嶙酸化 _-92- 本紙張尺度適用中國國家標準（CNS ) Α4規格（2l〇X297公釐） 1221482 經濟部中央標準局員工消費合作社印策 A7 B7 五、發明説明（90 ) 聯結子複體，上述經Κρη I和BamHI消化且純化的PCR產物 (參看D節），及經Nhe I和BamHI消化過的媒體pAMG22_His 之A片段吸連接在一起。將連接物用electroporation以製造商的程序轉形到大腸样菌宿主GM120内。選擇出菌株，分離出質體DNA並進行DNA定序以查證muOPG-HEK[22_401]基因的DNA序列。DNA定序顯露出自然muOPG序列中的假基因突變，其導致muOPG多肽的丙胺酸-45變成蘇胺酸之單一胺基酸變化。用類似C節中所述方法測定載有重組pAMG21質體的 GM120細胞之重組muOPG-HEK[22_401]之表現，不過，其中不添加合成自動謗導劑，而是添加IPTG到0.4 mM最後濃度以達到謗導作用。Oligo # 1331-92 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, -GAT CCT TAT TTT TGA GTT GAT TCA CTG TTT CCG GAA GA_3, (SEQ ID NO: 90) P. Human OPG met "27-1851, met "27-1891, met" 27-1941 The DNA sequences encoding the N · terminal methionine and amino acids 27 to 185, 27 to 189, or 27 to 194 contained in the human OPG polypeptide are placed as described below Prokaryotic • 90- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (21〇'〆297 mm) 1221482 A7 B7 V. Description of invention (88) Under the control of lux PR promoter gene contained in pAMG21 . The phosphorylated oligonucleotide linkers "huOPG_185 linker", "huOPG-189 linker" or "huOPG-194 linker" each containing Nde I and BamHI sticky ends (see section 0) And the decomposed plastid pAMG21-huOPG-met [27-401] digested with KpnI and NdeI endonucleases ~ 407 bp B fragment (see section 0), inserted in a guided way using standard methods The plastid PAMG21 · huOPG-met [27-401] is located between the κρηΙ and BamHI sites (see section J). Each linker was transformed into E. coli host 393 using electroporation according to the manufacturer's procedure. Select a strain, isolate plastid DNA, and perform DNA sequencing to verify the DNA sequence of the huOPG-met [27-185], huOPG-met [27-189], or huOI> G-met [27-194] gene . The performance of recombinant huOPG-met [27-185], huOPG-met [27_189], and huOPG-met [27-194] transformed into recombinant pAMG21 plastids transformed into 393 cells was determined using the method described in Section C. Q. Rat OPG metr27-401UP33E, G36S, A45P) Printed by the Central Standards Bureau of the Ministry of Economic Affairs ΗConsumer Cooperation 衽 (Please read the precautions on the back before filling this page) The N-terminal methionine and The DNA sequences of amino acids 27 to 48 and amino acid residues 49 to 401 contained in murine OPG were placed under the control of the lux PR promoter gene contained in prokaryotic expression media pAMG21 as described below. The purified pAMG21-huOPG-met [27-401] DNA (see section J) was cleaved with Aatll and κηΙ endonucleases and a ~ 1075 bp Β fragment was isolated from the agarose gel using standard recombinant DNA methods . In addition, pAMG21-muOPG-met [22-401] DNA (see section D) was digested with κρηΙ and BamHI endonucleases and ~ 1064 bp B fragments were isolated as described above. The standard recombinant DNA method will be used to isolate ~ 1075 bp -91 containing Aatll & κρηΙ sticky ends-This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 2 48 1X 2 12 A7 B7_ V. Description of the invention (89) pAMG21-huOPG-met [27-401] P-gen fragment (see above), ~ 1064 bp pAMG21-muOPG-met [22-401] restriction fragment containing the sticky ends of κρηΙ and BamHI A ~ 5043 bp restriction fragment containing Aatll and BamHI sticky ends and corresponding to the nucleic acid sequence of pAMG21 between Aatll and BamHI was ligated. The linker was transformed into E. coli host 393 via electroporatino using the manufacturer's procedure. Strains were selected and the presence of recombinant inserts in plastids was verified using standard DNA methods. muOPG-27_401 (P33E, G36S, A45P) gene. In muOPG, proline-33 becomes glutamic acid-33, glycine-36 becomes serine-36, and alanine-45 becomes proline-45. The amino acid changes are from muOPG residue 2 7 To 4 8 are facilitated by substitution of huOPG residues 2 7 to 48. The performance of recombinant muOPG_met [27-401] (P33E'G36S'A45P) of transformed 393] cells carrying recombinant pAMG21 plastid was determined by the method described in Section C. R. Rat OPG met-lvs- (his) 7-ala-ser- (asp) i-lys [22-401] (A45T) Printed by the Consumers ’Cooperative Bureau of the Ministry of Economic Affairs of the Central Government Bureau (please read the note on the back first) Please fill in this page for further information.) Encode the N-terminal His tag and enterokinase recognition sequence (from NH2 to COOH) of the mouse OPG polypeptide: Met-Lys-His-His-His-His-His-His-His- Ala_Ser-Asp_Asp-Asp-Asp-Lys (= HEK) 'The DNA sequences of amino acids 22 to 401 are placed under the control of the Ps4 promoter gene regulated by the lat-repressor as described below. PAMG22-His (see section A) was digested with Nhe I and BamHI endonucleases and large fragments (fragment A) were isolated from the agarose gel using standard recombinant DNA methods. Oligonucleotides # 1282-91 and # 1282-92 were separately phosphorylated and formed into oligonucleotide-linked complexes by the methods described above (see section B). Standard recombinant DNA methods will be used for acidification between oligonucleotides # 1282-91 and # 1282-92 and containing Nhe I and κρηΙ sticky ends _-92- This paper applies Chinese National Standards (CNS) Α4 size (210 × 297 mm) 1221482 Consumer Cooperative Cooperative Imprint A7 B7 of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention (90) Linker complex, the PCR product digested and purified by κρη I and BamHI (see section D) ), And the A fragment of pAMG22_His media digested by Nhe I and BamHI was sucked together. The linker was transformed into the coliform host GM120 using the manufacturer's procedure using electroporation. The strain was selected, and the plastid DNA was isolated and sequenced to verify the DNA sequence of the muOPG-HEK [22_401] gene. DNA sequencing revealed a pseudogene mutation in the natural muOPG sequence, which caused a change in the alanine-45 of the muOPG polypeptide to a single amino acid of threonine. The performance of recombinant muOPG-HEK [22_401] of GM120 cells containing recombinant pAMG21 plastid was measured by a method similar to that described in Section C. However, instead of adding a synthetic autodefense agent, IPTG was added to a final concentration of 0.4 mM to To achieve defamatory effects.

Oligo # 1282-91 5f-CTA GCG ACG ACG ACG ACA AAG AAA CTC TGC CTC CAA AAT ACC TGC ATT ACG ATC CGG AAA CTG GTC ATC AGC TGC TGT GTG AT A AAT GTG CTC CGG GTA C-3f (SEQ ID NO ： 91) Oligo # 1282-92 5f-CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT CCG GAT CGT AAT GCA GGT ATT TTG GAG GCA GAG TTT CTT TGT CGT CGT CGT CG-3丨（SEQ ID NO : 92) S .人類 OPG met-arg-ply-ser-fhis)422-4011 設計八種寡核苷酸（1338-09至1338-16，如下方者）來產生爲重疊，雙股DNA形式的175鹼片段。將該等寡核甞酸配對，連接，並使用Y和V寡核甞酸作爲PCR引子以產生大量 -93- ^紙張尺度適用中國國家標準(CNS ) A4規格（21〇Χ297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝· 訂 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（91 ) 的175鹼片段。將最後PCR基因產物用核酸内切限制酶Clal 和ΚρηΙ消化而得到取代人類OPG所含N-端28密碼子之片段。將經Clal和ΚρηΙ消化過的PCR產物插置到也用Clal和 ΚρηΙ切過的pAMG21-huOPG[27-4〇l]之内。將連接DNA轉形到勝任的大腸桿菌菌株393宿主細胞内。針對產生重組蛋白質產物及擁有具有正碓核甞酸序列的基因融合物之能力篩選菌株。用50毫升搖動機燒瓶斫究來測定蛋白質表現水平。對全細胞溶裂物和聲波丸粒用鼠抗-OPG抗體以考馬斯染色PAGE凝膠和西方氏分析進行構成物表現。導致大包涵體和蛋白質的形成之huOPG Met-Arg-Gly_Ser(His)6 [22-401] 表現係集中在不溶性（丸）部份。 1338-09 ACA AAC ACA ATC GAT TTG ATA CTA GA (SBQ ID NO：93) 1338-10 TTT GTT ΤΤΑ ACT ΑΑΤ T7VA AGG AGG AAT AAA ATA TGA GAG GAT GGC ATC AC (SBQ ID NO:94) 1338-11 CAT CAC CAT CAC GAA ACC TTC CCG CCG AAA TAC CTG CAC TAC GAC GAA GA (SBQ ID NO:95) 1338-12 AAC CTC CCA CCA GCT GCT GTG CGA CAA ATG CCC GCC GGG TAC CCA AAC A (SEQ ID NO:96) 1338-13 TGT TTG GGT ACC CGG CGG GCA TTT GT (SBQ ID NO：97) 1338-14 CGC ACA GCA GCT GGT GGG AGG TTT CTT CGT CGT AGT GCA GGT ATT TCG GC (SEQ ID NO：98) -94· 本紙張尺度適用中國國家榡準（CNS ) A4規格（210X297公釐）~~~ ---------*^------1T------ (請先閲讀背面之注意事項再填寫本頁) 1221482 經濟部中央標準局員工消費合作社印策 A7 _____B7 五、發明説明（92 ) 1338-15 GGG AAG GTT TCG TGA TGG TGA TGG TGA TGC GAT CCT CTC ΑΤΑ TTT TAT T (SEQ ID NO:99) 1338-16 CCT CCT TTA ATT AGT TAA AAC AAA TCT AGT ATC AAA TCG ATT GTG TTT GT (SE)Q ID NO： 100) I·人類 OPG met-lysf22-4011 和metnvshUldOl] 爲構成人類OPG[22_401]的met_lys和met-(lys)3形式，乃設計重疊寡核苷酸以添加恰當數目的離胺酸殘基。每一構成物的兩個寡核甞酸係經設計成重疊者，以使兩次PCR即可產生最後產物。第一次PCR反應的模版爲含有人類OPG 22-401 基因的質體DNA製備物。第一次PCR係添加離胺酸殘基。第二次PCR使用第一次的產物並將序列加回到第一限制部位， ClaI〇將最後PCR基因產物用核酸内切限制酶Clal和ΚρηΙ消化，取代人類OPG的Ν-端28密碼子，然後將其連接到也已用該兩核酸内切限制酶消化過的質體pAMG21-hu OPG-[27-401] 之内。將連接後的DNA轉形到勝任的大腸样菌菌株393宿主細胞内。針對產生重組蛋白質產物及擁有具有正確核甞酸序列的基因融合體之能力篩選菌株。用50毫升搖動器燒瓶研究來測定蛋白質表現水平。對全細胞溶裂物和聲波沈丸用鼠抗· OPG抗體考馬斯染色PAGE凝膠和西方氏分析進行構成物表現分析。任一構成物皆未有可偵檢的蛋白質表現水平且未能看到包涵體。以DNA定序確定DNA序列。製備Met_LyshuOPG[22-401]的寡核甞酸引子： -95- 本紙張尺度適用中國國家標準（CNS〉A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝· 訂 1221482 A7 B7 五、發明説明（93 ) 1338-17 ACA AAC ACA ATC GAT TTG ATA CTA GAT TTG TTT ΤΑΑ CTA ATT AAA GGA GGA ΑΤΑ AAA TG ( SEQ ID NO: 101) 1338-18 CTA ATT AAA GGA GGA ΑΤΑ i AAT ATC (SEQ ID NO:102)Oligo # 1282-91 5f-CTA GCG ACG ACG ACG ACA AAG AAA CTC TGC CTC CAA AAT ACC TGC ATT ACG ATC CGG AAA CTG GTC ATC AGC TGC TGT GTG AT A AAT GTG CTC CGG GTA C-3f (SEQ ID NO: 91 ) Oligo # 1282-92 5f-CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT CCG GAT CGT AAT GCA GGT ATT TTG GAG GCA GAG TTT CTT TGT CGT CGT CGT CG-3 丨 (SEQ ID NO: 92) S Human OPG met-arg-ply-ser-fhis) 422-4011 Eight oligonucleotides (1338-09 to 1338-16, below) were designed to produce 175 base fragments in the form of overlapping, double-stranded DNA. These oligonucleotides were paired, ligated, and Y and V oligonucleotides were used as PCR primers to generate a large amount of -93- ^ Paper size is applicable to China National Standard (CNS) A4 specification (21 × 297 mm) (please Read the notes on the back before filling out this page) Binding and ordering 1221482 A7 B7 Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs V. 175 alkali fragment of the invention description (91). The final PCR gene product was digested with the endonucleases Clal and KpηI to obtain a fragment replacing the N-terminal 28 codon contained in human OPG. The PCR product digested with Clal and KρηI was inserted into pAMG21-huOPG [27-4〇l], which was also cut with Clal and KρηΙ. The ligated DNA was transformed into a competent E. coli strain 393 host cell. Strains were screened for their ability to produce recombinant protein products and possess gene fusions with orthonucleotide sequences. A 50 ml shaker flask was investigated to determine the level of protein performance. The whole-cell lysates and sonic pellets were constructed using mouse anti-OPG antibody using Coomassie stained PAGE gel and Western analysis. The huOPG Met-Arg-Gly_Ser (His) 6 [22-401] that leads to the formation of large inclusion bodies and proteins is concentrated in the insoluble (pellet) part. 1338-09 ACA AAC ACA ATC GAT TTG ATA CTA GA (SBQ ID NO: 93) 1338-10 TTT GTT ΤΤΑ ACT ΑΑΤ T7VA AGG AGG AAT AAA ATA TGA GAG GAT GGC ATC AC (SBQ ID NO: 94) 1338-11 CAT CAC CAT CAC GAA ACC TTC CCG CCG AAA TAC CTG CAC TAC GAC GAA GA (SBQ ID NO: 95) 1338-12 AAC CTC CCA CCA GCT GCT GTG CGA CAA ATG CCC GCC GGG TAC CCA AAC A (SEQ ID NO: 96) 1338-13 TGT TTG GGT ACC CGG CGG GCA TTT GT (SBQ ID NO: 97) 1338-14 CGC ACA GCA GCA GCT GGT GGG AGG TTT CTT CGT CGT AGT GCA GGT ATT TCG GC (SEQ ID NO: 98) -94 · 本Paper size applies to China National Standard (CNS) A4 (210X297 mm) ~~~ --------- * ^ ------ 1T ------ (Please read the Please fill in this page again) 1221482 Imprint A7 _____B7 by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs V. Invention Description (92) 1338-15 GGG AAG GTT TCG TGA TGG TGA TGG TGA TGC GAT CCT CTC ΑΑΑ TTT TAT T (SEQ ID NO: 99) 1338-16 CCT CCT TTA ATT AGT TAA AAC AAA TCT AGT ATC AAA TCG ATT GTG TTT GT (SE) Q ID NO: 100) I. Human OPG met-lysf22-4011 and metnvshUldOl] constitute human OPG [ 22_401] 's met_lys and met- (lys) 3 forms are designed to overlap oligonucleotides to add the appropriate number of lysine residues. The two oligonucleotides of each construct are designed to overlap so that two PCRs can produce the final product. The template for the first PCR reaction was a plastid DNA preparation containing the human OPG 22-401 gene. In the first PCR, lysine residues were added. The second PCR uses the first product and adds the sequence back to the first restriction site. ClaI0 digests the final PCR gene product with the endonucleases Clal and κηΙ to replace the N-terminal 28 codon of human OPG. It was then ligated into pAMG21-hu OPG- [27-401], which had also been digested with the two endonucleases. The ligated DNA was transformed into a competent coliform-like strain 393 host cell. Strains were screened for their ability to produce recombinant protein products and possess gene fusions with the correct nucleotide sequence. A 50 ml shaker flask study was used to determine protein expression levels. The whole cell lysate and sonic sinking pill were analyzed for structure expression using a mouse anti-OPG antibody Coomassie stained PAGE gel and Western analysis. None of the constituents had detectable levels of protein expression and no inclusion bodies were visible. The DNA sequence is determined by DNA sequencing. Preparation of oligonucleotide primers for Met_LyshuOPG [22-401]: -95- This paper size applies to Chinese national standards (CNS> A4 size (210X297 mm) (Please read the precautions on the back before filling this page). Binding 1221482 A7 B7 V. Description of the Invention (93) 1338-17 ACA AAC ACA ATC GAT TTG ATA CTA GAT TTG TTT ΤΑΑ CTA ATT AAA GGA GGA ΑΤΑ AAA TG (SEQ ID NO: 101) 1338-18 CTA ATT AAA GGA GGA ΑΑΑ i AAT ATC (SEQ ID NO: 102)

TGA AAGTGA AAG

CTT TTC CTC CAA 1338-20 TGT TTG GGT ACC CGG CGG ACA TTT ATC ACA C (SEQ ID NO: 103) Oligonucleotide primers to prepare Met-(Lys>3_huOPG[22-401]: 1338-17 ACA AAC ACA ATC GAT TTG ATA CTA GAT TTG TTT TAA CTA ATT AAA GGA GGA ATA AAA TG (SEQ ID NO: 104) (請先閱讀背面之注意事項再填寫本頁) 裝· 訂CTT TTC CTC CAA 1338-20 TGT TTG GGT ACC CGG CGG ACA TTT ATC ACA C (SEQ ID NO: 103) Oligonucleotide primers to prepare Met- (Lys > 3_huOPG [22-401]: 1338-17 ACA AAC ACA ATC GAT TTG ATA CTA GAT TTG TTT TAA CTA ATT AAA GGA GGA ATA AAA TG (SEQ ID NO: 104) (Please read the precautions on the back before filling out this page)

AAGAAG

CTT TTC 經濟部中央標準局員工消費合作社印製 1338-19 CTA ATT AAA GGA GGA ATA AAA TGA CTC CAA AAT ATC (SEQ 工D NO: 105 1338-20 TGT TTG GGT ACC CGG CGG ACA TTT ATC ACA C (SEQ ID NO: 106) U.人類和鼠OPG丨22_4011/Fc融合體構成四種OPG-Fc融合體，其中係將人類IgGl的F c區融合到人類或鼠促骨堆積肽胺基酸2 2到401的N -端（稱爲Fc/OPG [22-401])或融合到C-端（稱爲〇PG[22_401]/Fc)。Fc融合體係用實施例7中所述融合媒體pFc-A3構成者。所有的融合基因都是用標準PCR技術構成的。PCR反應的模版爲含有標的基因的質體製備物。設計重疊寡核苷酸以 96- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（94 ) 將一基因的C -端部份與另一基因的N端部份組合起來。此方法可將兩基因在實施恰當的PCR反應後以正確的讀碼融合在一起。初始時係將每一基因的”融合”寡核甞酸與載著標的基因的媒體所用之通用引體一起放入PCR反應中。互補" 融合π寡核甞酸則與一通用引子一起用來PCR另一基因。於此第一 PCR反應結束時，得到兩個別的產物，每一基因各含其融合部位，造成足夠的重疊以驅使第二回PCR及造成所欲融合體。於第二回PCR中，第一回的兩種產物係與通用引子組合且透過重疊區而產生全長度融合DNA序列。將最後PCR基因產物用核酸内切限制酶Xbal與BamHI消化後，連接到也已用該兩種相同核酸内切限制酶消化過的媒體pAMG21内。將連接後的DNA轉形到勝任的大腸桿菌菌株 393宿主細胞内。針對產生重組蛋白質產物及擁有具正確核甞酸序列的基因融合體之能力篩選菌株。用50毫升搖動機燒瓶研究測定蛋白質表現水平。全細胞溶裂物，聲波丸，和上澄液都經考馬斯染色PAGE凝膠和用鼠抗-OPG抗體進行的西方氏分析予以分析融合體的表現。CTT TTC Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 1338-19 CTA ATT AAA GGA GGA ATA AAA TGA CTC CAA AAT ATC (SEQ ID NO: 105 1338-20 TGT TTG GGT ACC CGG CGG ACA TTT ATC ACA C (SEQ ID NO: 106) U. Human and murine OPG 丨 22_4011 / Fc fusions constitute four types of OPG-Fc fusions, in which the F c region of human IgG1 is fused to human or murine osteotrophic peptide amino acid 2 2 to The N-terminus of 401 (referred to as Fc / OPG [22-401]) or fused to the C-terminus (referred to as PG [22_401] / Fc). The Fc fusion system uses the fusion media pFc-A3 as described in Example 7. Constructor. All fusion genes are constructed using standard PCR technology. The template of the PCR reaction is a plastid preparation containing the target gene. The overlapping oligonucleotides are designed to 96- this paper scale applies Chinese National Standard (CNS) A4 Specifications (21 × 297 mm) 1221482 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 5. Invention Description (94) Combine the C-terminal part of one gene with the N-terminal part of another gene. This The method can fuse the two genes with the correct reading code after performing the proper PCR reaction. The "fusion" oligonucleotide of each gene is put into a PCR reaction with the universal primers used in the media carrying the target gene. The complementary " fusion π oligonucleotide is used with one universal primer to PCR the other Gene. At the end of the first PCR reaction, two other products are obtained, each gene contains its fusion site, causing sufficient overlap to drive the second PCR and the desired fusion. In the second PCR The first two products were combined with universal primers and produced full-length fusion DNA sequences through overlapping regions. The final PCR gene product was digested with endonucleases Xbal and BamHI, and ligated to these two The same endonuclease digested media pAMG21. The ligated DNA is transformed into competent E. coli strain 393 host cells. Ability to produce recombinant protein products and gene fusions with the correct nucleotide sequence Strains were screened. The protein expression levels were determined using a 50 ml shaker flask study. Whole cell lysates, sonic pills, and supernatant were all Coomassie-stained PAGE gels and anti-OPG antibody The performance of the fusion was analyzed by Western analysis performed by the system.

Fc/huQPG[22-401] 在考馬斯染色PAGE凝膠上和西方氏吸潰上偵檢Fc/huOPG [22-401]融合肽的表現。該細胞具有非常大的包涵體，且其大部份的產物係在不溶（丸）部份内。用下列引子來構成此 0PG_Fc融合體·· 1318-48Fc / huQPG [22-401] was used to detect the expression of Fc / huOPG [22-401] fusion peptide on Coomassie stained PAGE gel and Western blot. The cells have very large inclusion bodies, and most of their products are in the insoluble (pellet) portion. The following primers were used to construct this 0PG_Fc fusion ... 1318-48

CAG CCC GGG TAA AAT GGA AAC GTT TCC TCC AAA -97- 本紙張尺度適用中國國家標準（CNS ) A4規格（210Χ297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝· 訂 1221482 A7 B7 五、發明説明（95 ) ATA TCT TCA TT (SEQ ID NO ： 107) 1318-49 CGT TTC CAT TTT ACC CGG GCT GAG CGA GAG GCT CTT CTG CGT CT (SEQ ID NO : 108)CAG CCC GGG TAA AAT GGA AAC GTT TCC TCC AAA -97- This paper size applies to China National Standard (CNS) A4 specifications (210 × 297 mm) (Please read the precautions on the back before filling this page) Binding and ordering 1221482 A7 B7 V. Description of the invention (95) ATA TCT TCA TT (SEQ ID NO: 107) 1318-49 CGT TTC CAT TTT ACC CGG GCT GAG CGA GAG GCT CTT CTG CGT CT (SEQ ID NO: 108)

Fc/muQPG[22-401] 在考馬斯染色凝膠和西方氏吸潰上偵檢融合肽的表現。細胞具有非常大的包涵體，且大部份的產物都在不溶（丸）部份内。使用下列引子來構成此OPG-Fc融合物： 1318-50 CGC TCA GCC CGG GTA AAA TGG AAA CGT TGC CTC CAA AAT ACC TGC (SEQ ID NO : 109) 1318-51Fc / muQPG [22-401] was used to detect the expression of fusion peptides on Coomassie gel and Western blot. The cells have very large inclusions, and most of the products are in the insoluble (pellet) fraction. The following primers were used to construct this OPG-Fc fusion: 1318-50 CGC TCA GCC CGG GTA AAA TGG AAA CGT TGC CTC CAA AAT ACC TGC (SEQ ID NO: 109) 1318-51

CCA TTT TAC CCG GGC TGA GCG AGA GGC TCT TCT GCG TGT (SEQ ID NO ·· 110) muOPGr22-401]/Fc 經濟部中央標準局員工消費合作社印装 (請先閱讀背面之注意事項再填寫本頁) 在考馬斯染色凝膠和西方氏吸潰上偵檢融合肽的表現。重組產物的量少於在N端具有Fc區的OPG融合蛋白質。未偵檢到明顯的包涵體。大部份的產物顯示在不溶（丸）部份内。使用下列引子來構成OPG-Fc融合體： 1318-54 CAA AAT AAG CTG CTT AGC TGC AGC TGA ACC AAA ATC (SEQ ID NO : 111) 1318-55 -98- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（96 )CCA TTT TAC CCG GGC TGA GCG AGA GGC TCT TCT GCG TGT (SEQ ID NO · · 110) muOPGr22-401] / Fc Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the precautions on the back before filling this page) Detection of fusion peptides on Coomassie gels and Western blots. The amount of the shuffle product was less than the OPG fusion protein having an Fc region at the N-terminus. No obvious inclusions were detected. Most of the product appears in the insoluble (pellet) portion. The following primers were used to construct the OPG-Fc fusion: 1318-54 CAA AAT AAG CTG CTT AGC TGC AGC TGA ACC AAA ATC (SEQ ID NO: 111) 1318-55 -98- This paper standard applies Chinese National Standard (CNS) A4 Specifications (210X 297 mm) 1221482 A7 B7 V. Description of the invention (96)

CAG CTG CAG CTA AGC AGC TTA TTT TCA CGG ATT G (SEQ ID NO : 112) huOPG「22-4011/Fc 在考馬斯染色凝膠上未偵檢到融合肽的表現，雖則含有微弱的西方氏陽性信號。使用下列引子來製備此OPG-Fc融合體： 1318-52 AAA AAT AAG CTG CTT AGC TGC AGC TGA ACC AAA ATC (SEQ ID NO ： 113) 1318-53 CAG CTG CAG CTA AGC AGC TTA TTT TTA CTG ATT GG (SEQ ID NO : 114) V·人類 OPG met 丨 22-4011_Fc 融合體（P25A) 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 此構成物將在位置25的脯胺酸變成丙胺酸的胺基酸變化 (P25A)與huOPG met[22-401]-Fc融合體組合在一起。將質體用核酸限制酶Clal和ΚρηΙ消化以脱除基因的N-端2 8個密碼子，並將所得小（少於200鹼對）片段以凝膠純化。然後將含有脯胺酸成爲丙胺酸變化的此片段連接到已用該兩種核酸内切酶消化過的質體pAMG21-lmOPG[22-401]-Fc融合體之内。將連接好的DNA轉形到勝任的大腸桿菌菌株393宿主細胞内。針對產生重組蛋白質產物及擁有具有正確核苷酸序列的基因融合體之能力篩選菌株。用50毫升搖動器燒瓶研究來測定蛋白質表現水平。經由考馬斯染色PAGE凝膠及用 -99- 本紙張尺度適用中國國家榡準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7__ 五、發明説明（97 ) 鼠抗-OPG抗體進行西方氏分析以分析全細胞溶裂物和聲波丸的構成物表現。在考馬斯染色PAGE凝膠和西方氏吸潰上偵檢融合肽的表現水平。蛋白質係在不溶（丸）部份内。細胞具有大包涵體。 W.人類〇PG metr22-4011 (P25A) 編碼其位碼2 5處的脯胺酸被丙胺酸取代的人類OPG所含 N-端甲硫胺酸和胺基酸22至401且係在原核生物表現媒體 pAMG21所含lux PR啓動基因控制之下的DNA序列係以下述構成的：將合成寡核苷酸#1289-84和1289-85配對形成具有 Xbal和ΚρηΙ黏狀末端的寡核甞酸聯結子複體。該合成聯結子複體係利用最適的大腸桿菌密碼子並編碼Ν-端甲硫胺酸。該聯結子也包含一個在原序列中不存在的Spel限制部位。將該聯結子複體用標準方法導引地插置在PAMG21-huOPG-22-401中的Xbal與ΚρηΙ部位之間。將連接混合物轉形導到大腸桿菌宿主GM221内。先篩選生產重組蛋白質的菌株。從陽性菌株分離出質體DNA並進行DNA定序以查證 HuOPG-Met[22-401] (Ρ25Α)基因的DNA序列。使用下列寡核甞酸來產生Xbal-Kpnl聯結子：CAG CTG CAG CTA AGC AGC TTA TTT TCA CGG ATT G (SEQ ID NO: 112) huOPG "22-4011 / Fc No fusion peptide was detected on the Coomassie gel, although it contained a weak Western positive Signal. The following primers were used to make this OPG-Fc fusion: 1318-52 AAA AAT AAG CTG CTT AGC TGC AGC TGA ACC AAA ATC (SEQ ID NO: 113) 1318-53 CAG CTG CAG CTA AGC AGC TTA TTT TTA CTG ATT GG (SEQ ID NO: 114) V · Human OPG met 丨 22-4011_Fc Fusion (P25A) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) The amino acid change (P25A) of proline to alanine at position 25 was combined with the huOPG met [22-401] -Fc fusion. The plastids were digested with the nucleic acid restriction enzymes Clal and κηΙ to remove the N -Terminal 2 8 codons, and the resulting small (less than 200 base pairs) fragments were gel purified. This fragment containing the change in proline to alanine was then ligated to the two endonucleases that had been digested Into the plastid pAMG21-lmOPG [22-401] -Fc fusion. Transfer the ligated DNA Shaped into competent E. coli strain 393 host cells. Screen strains for their ability to produce recombinant protein products and possess gene fusions with the correct nucleotide sequence. Use a 50 ml shaker flask study to determine protein performance levels. Via Kaoma Stained PAGE gel and use -99- This paper size is applicable to China National Standards (CNS) A4 specifications (210X297 mm) 1221482 Printed by A7 B7 of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (97) -OPG antibody was subjected to Western analysis to analyze the performance of whole cell lysates and sonic pills. Detection of fusion peptide performance on Coomassie PAGE gels and Western blots. Proteins were insoluble (pill ). Cells have large inclusion bodies. W. Human OPG metr22-4011 (P25A) encodes N-terminal methionine and The DNA sequences of amino acids 22 to 401 and under the control of the lux PR promoter gene contained in the prokaryotic expression media pAMG21 were constructed as follows: the synthetic oligonucleotides # 1289-84 and 1289-85 were paired Chang acid oligonucleotide having Xbal and ΚρηΙ sticky and blunt ends of the linker duplexes. This synthetic linker complex utilizes the optimal E. coli codon and encodes an N-terminal methionine. The linker also contains a Spel restriction site that was not present in the original sequence. This linker complex was guidedly inserted between the Xbal and KKn sites in PAMG21-huOPG-22-401 by standard methods. The ligation mixture was transformed into E. coli host GM221. The strains producing recombinant protein are first screened. Plastid DNA was isolated from the positive strain and DNA sequencing was performed to verify the DNA sequence of the HuOPG-Met [22-401] (P25A) gene. The following oligonucleotides were used to generate Xbal-Kpnl linkers:

Oligo #1289-84 5f-CTA GAA GGA GGA ΑΤΑ ACA TAT GGA AAC TTT TGC TCC AAA ATA TCT TCA TTA TGA TGA AGA AAC TAG TCA TCA GCT GCT GTG TGA TAA ATG TCC GCC GGG TAC-3 丨(SEQ ID NO : 115)Oligo # 1289-84 5f-CTA GAA GGA GGA ΑΤΑ ACA TAT GGA AAC TTT TGC TCC AAA ATA TCT TCA TTA TGA TGA AGA AAC TAG TCA TCA GCA GCT GCT GTG TGA TAA ATG TCC GCC GGG TAC-3 丨 (SEQ ID NO: 115 )

Oligo #1289-85 -100- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） ---------------IT----- (請先閲讀背面之注意事項再填寫本頁) 1221482 A7 B7 五、發明説明（98 ) 5f-CCG GCG GAC ATT TAT CAC ACA GCA GCT GAT GAC TAG TTT CTT CAT CAT AAT GAA GAT ATT TTG GAG CAA AAG TTT CCA TAT GTTATTCCTCCTT-3,（SEQ ID NO : 116) X.人類OPG met『22-4011 (P26A)和（P26D) 將編碼其2 6位置的脯胺酸被丙胺酸取代的人類OPG所含 N-端甲硫胺酸和胺基酸2 2至401且係在原核生物表現媒體 pAMG21所含lux PR啓動基因控制之下的DNA序列係以下文所述製備的：將合成寡核苷酸#1289-86和1289-87配對形成具有黏狀末端的寡核甞酸聯結子複體。該合成聯結子複體係利用最適的大腸桿菌密碼且編碼著一 N-端甲硫胺酸。用標準方法將該聯結子複體導引地插置於pAMG21 -huOPG[22-401](P25A)所含Xbal與Spel兩部位之間。將連接混合物轉形導到大腸样菌宿主GM221内。先篩選生生產重組蛋白質的菌株。從陽性菌株分離出質體DNA並進行DNA定序以查證 huOPG-met[22-401] (P26A)基因的DNA序列。被定序過的菌株之一經發現在26位置處的脯胺酸被天冬胺酸而非丙胺酸所取代，而將此株命名爲huOPG_met[22-401](P26D)。使用下列寡核甞酸來產生Xbal-Spel聯結子：Oligo # 1289-85 -100- This paper size applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) --------------- IT ----- (please first Read the notes on the back and fill out this page) 1221482 A7 B7 V. Invention Description (98) 5f-CCG GCG GAC ATT TAT CAC ACA GCA GCT GAT GAC TAG TTT CTT CAT CAT AAT GAA GAT ATT TTG GAG CAA AAG TTT CCA TAT GTTATTCCTCCTT -3, (SEQ ID NO: 116) X. Human OPG met "22-4011 (P26A) and (P26D) N-terminal methyl sulfur contained in human OPG whose proline at position 2 6 is replaced by alanine DNA sequences of amino acids and amino acids 22 to 401 and under the control of the lux PR promoter gene contained in the prokaryotic expression media pAMG21 were prepared as follows: synthetic oligonucleotides # 1289-86 and 1289 -87 paired to form a complex of oligonucleotide linkers with sticky ends. The synthetic linker complex uses the optimal E. coli code and encodes an N-terminal methionine. The linker complex was guidedly inserted between the two parts Xbal and Spel contained in pAMG21-huOPG [22-401] (P25A) by standard methods. The ligation mixture was transformed into the coliform host GM221. First, strains that produce recombinant protein are screened. Plastid DNA was isolated from the positive strain and DNA sequencing was performed to verify the DNA sequence of the huOPG-met [22-401] (P26A) gene. One of the sequenced strains was found to have proline at position 26 replaced by aspartic acid instead of alanine, and this strain was named huOPG_met [22-401] (P26D). The following oligonucleotides were used to generate Xbal-Spel linkers:

Oligo #1289-86 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 5'-CTA GAA GGA GGA ΑΤΑ ACA TAT GGA AAC TTT TCC TGC TAA ATA TCT TCA TTA TGA TGA AGA AA-3,（SEQ ID NO : 117)Oligo # 1289-86 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) 5'-CTA GAA GGA GGA ΑΑΑ TCA GAT AAC TTT TCC TGC TAA ATA TCT TCA TTA TGA TGA AGA AA-3, (SEQ ID NO: 117)

Oligo #1289-87Oligo # 1289-87

5'-CTA GTT TCT TCA TCA TAA TGA AGA TAT TTA GCA GGA AAA -101 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（99 ) GTT TCC ATA TGT TAT TCC TCC TT-3,（SEQ ID NO : 118) Y.人類OPG met『22-1941 (P25A) 將編碼其2 5位置處的脯胺酸被丙胺酸取代的人類OPG所含N-端甲硫胺酸和胺基酸22至194且係在原核生物表現媒體pAMG21所含lux PR啓動基因控制之下的DNA序列依下述構成：將質體 pAMG21-huOPG[27_194]和 pAMG21-huOPG [22-401](P25A)分別用ΚρηΙ和BamHI核酸内切酶予以消化。從pAMG21-huOPG[27-194]分離出 450 bp片段且從 pAMG21-huOPG[22-401](P25A)分離出6.1 kbp片段。將這些片段連接在一起並轉形導到大腸样菌宿主GM221内。先篩選出生產重組蛋白質的菌株。從陽性菌株分離出質體DNA且定序 DNA以查證huOPG-Met[22-194](P25A)基因的 DNA序列。實施例9 OPG單體的缔合使用經處理成過度表現muOPG[22-401]的CHO細胞產生調理培養基以供使用兔子多株抗-OPG抗體進行分泌重組OPG 分析所用。將一液份的調理培養基濃縮20-倍，然後以還原性和非還原性SDS-PAGE予以分析（圖15)。在還原性條件下，蛋白質像一 Mr 50-55 kd多肽一般移動，如同在將成熟產物在其一或多個一致的N-鍵聯糖基化部位予以糖基化後所預測者。令人訝異者，在用非還原性SDS-PAGE分析相同的樣品時，大部份的蛋白質係依約1〇〇 kd多肽而移動，爲被還原蛋白質的兩倍尺寸。此外，還有較少量的Nr 50-55 kd 多肽。這種在SDS-PAGE上的移動樣式與OPG產物經由自由 -102- 本紙張尺度適用中國國家檩準（CNS ) A4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁)5'-CTA GTT TCT TCA TCA TAA TGA AGA TAT TTA GCA GGA AAA -101-This paper size applies to the Chinese National Standard (CNS) A4 (210X 297 mm) 1221482 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (99) GTT TCC ATA TGT TAT TCC TCC TT-3, (SEQ ID NO: 118) Y. Human OPG met "22-1941 (P25A) will encode the proline acid at position 2 5 by propylamine The DNA sequence of N-terminal methionine and amino acids 22 to 194 contained in acid-substituted human OPG and under the control of the lux PR promoter gene contained in prokaryotic expression media pAMG21 is as follows: plastid pAMG21 -huOPG [27_194] and pAMG21-huOPG [22-401] (P25A) were digested with κρηΙ and BamHI endonucleases, respectively. A 450 bp fragment was isolated from pAMG21-huOPG [27-194] and a 6.1 kbp fragment was isolated from pAMG21-huOPG [22-401] (P25A). These fragments were ligated together and transduced into the coliform host GM221. First, strains producing recombinant proteins were screened. Plastid DNA was isolated from the positive strain and sequenced to verify the DNA sequence of the huOPG-Met [22-194] (P25A) gene. Example 9 Association of OPG monomers CHO cells treated to overexpress muOPG [22-401] were used to generate a conditioning medium for use in rabbit recombinant anti-OPG antibodies for secretory recombinant OPG analysis. One aliquot of the conditioning medium was concentrated 20-fold and analyzed by reducing and non-reducing SDS-PAGE (Figure 15). Under reducing conditions, the protein moves like a Mr 50-55 kd polypeptide, as predicted by glycosylation of the mature product at one or more consistent N-linked glycosylation sites. Surprisingly, when the same sample was analyzed by non-reducing SDS-PAGE, most of the protein lines moved by about 100 kd polypeptide, which was twice the size of the reduced protein. In addition, there are smaller amounts of Nr 50-55 kd peptides. This mobile style on SDS-PAGE and OPG products pass freely -102- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) (Please read the precautions on the back before filling this page)

1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（100 ) 氫硫基氧化形成二體物之説法一致。預期的成熟OPG多肽含有23半胱胺酸殘基，其中18個預測有參與鏈間雙硫橋聯的形成，其中包括四個富含半胱胺酸的域（圖12A)。五個剩餘的C-端半胱胺殘基不涉及在二級結構中，此點可根據與其他TNFR族員的同系性而預測。整體而言，其中有淨非偶數的光胱胺酸殘基，且就形式上而言可能有至少一個殘基係自由者可以形成兩個OPG單體之間的分子間雙硫鍵。爲了有助於解開OPG動力學和單體缔合樣式，乃進行脈衝 -追踪標記研究。將表現muOPG[22-401]的CHO細胞依上文所述在含有35S甲硫胺酸和半胱胺酸的無血清培養基中代謝地標記30分鐘。在此期後，取出培養基，並換上完全培養基，其中含有約爲原來放射性胺基酸濃度約2,000-倍超量的水平之未標記甲硫胺酸和半胱胺酸。於添加後3 〇分鐘，1小時，2小時，4小時，6小時和1 2小時，經由脱除調理培養基而收取培養物，並製備調理培養基及吸附單層的溶裂物。依上述將培養物的培養基和細胞溶裂物予以澄清，然後依上述用抗-OPG抗體予以免疫沈澱。於洗滌過免疫沈澱物後，經由在非還原性SDS-PAGE緩衝液中煮沸使彼等釋出，再平分成兩半。於其中一半，將還原劑卢·氳硫基乙醇加到5%(v/v)最後濃度，同時將另一半保持在非還原性狀況下。依上文所述以SDS-PAGE分析兩組免疫沈澱物後，進行放射自顯術處理並曝光到底片。其結果示圖1 6中。經由還原性SDS-PAGE分析過的樣品示於下面兩格組中。於合成 -103- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ---------^-裝-- (請先閲讀背面之注意事項再填寫本頁) 訂 1221482 A7 B7 五、發明説明（101 ) 後，將OPG多肽迅速處理成稍微較大的多肽，其可能展現出 N-鍵聯糖基化改質形式。於約1-2小時後，細胞中的〇?&水平急劇下降，且同時出現於培養物上澄液中。此種現象似乎爲OPG隨時間從細胞經由媒體輸送到培養基中之結果，與 OPG爲天然分泌蛋白質的説法一致。分析在非還原性條件下的相同免疫沈澱物時揭露出在〇PG二體物形成與分泌到調理培養基中之間的關係（圖1 6，上面格）。於前面30-60分鐘内，OPG單體在細胞内經由明顯的糖基化反應處理，接著即二體物形成。隨著時間，大部份的〇PG單體都被形成爲二體物，其隨後即從細胞中消失。於合成後約6〇分開始，0PG 二體物即出現在調理培養基中，並隨實驗的持續而蓄積。在此期間之後，形成OPG二體物，其隨後即分泌到培養物培養基内。OPG單體則内則在細胞内隨時間以低水平保持著，且也有少量出現在培養基中。此似乎不足共價鍵結〇PG二體物分解的結果，而是次化學計算量的單體在細胞内產生及隨後分泌出所致。經濟部中央標準局員工消費合作社印製 ---------^-裴-- (請先閱讀背面之注意事項再填寫本頁) 從轉染CHO細胞重組產生的OPG主要爲二體物。爲了決定二體化是否爲OPG合成中的自然程序，我們分析可自然表現 OPG的細胞系之調理培養基。CTLL-2細胞系一種鼠類細胞毒性T淋巴細胞系（ATCC登錄號碼TIB_214)經發現可在一組組織和細胞系RNA中表現OPG mRNA。其中OPG轉錄本經發與從腎鑑別出，經選殖和定序過的2.5-3.0kbRNA相同且經發現其編碼著分泌分子。從CTLL-2細胞得到的調理培養基經西方氏吸潰分析顯示，若非全部也有大部份的分泌出之 -104- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X 297公釐） 1221482 經濟部中央標準局員工消費合作社印策 A7 B7 五、發明説明（1〇2) 〇PG蛋白質爲二體物（圖17)。此點可推測〇pG二體化和分泌不是人工在細胞系内過度表現所致，而可能是產物被表現性細胞產生時的主要產物形式。將正常和導入外來基因的小鼠組織物與血清進行分析以測疋在〇PG導入外來是基因小鼠内表現出的OPG分子之本質。由於老鼠OPG cDNA係在肝細胞控制要素之下表現者，因此使用對照小鼠和導入外來基因的小鼠之實質在非還原條件下所得萃取物進行分析（圖i 8)。於來自導入外來基因的小鼠而非對照小鼠的萃取物中，可順利地偵檢到〇PG二體物，以及化學計算量的單體。該〇PG二體物和單體物顯得與在經遺傳工程處理過的CHO細胞中表現的重組鼠蛋白質相同。此點強烈地推測OPG二體物確實是基因產物的自然形式，且可能爲關鍵性活性成份，將得自對照小鼠和導入外來基因小鼠的血清樣品也用西方氏吸潰分析進行類似的分析。於對照小鼠中，大部份的OPG蛋白質係以二體物形式移動，同時也偵檢出少量的單體物。此外，也偵檢出明顯量的較大型OPG相關蛋白質’其以與預測的共價键聯二體物^一致的相對分子量移動。因此，重組OPG在OPG導入外來基因小鼠内主要係表現成二體物蛋白質，且該二體物形式可能爲OPG小鼠内的骨質石化表現型之基礎。OPG重組蛋白質也可以存在於更高分子量的”參體物"形式之中。於了測定OPG所含的5個C_端半胱胺酸殘基是否在同元二體化中具有作用，乃依上文所述使用the QuickChange™ Site-Directed Mutagenesis Kit(Stratagene，San Diego，CA)將 -105 - 本紙張尺度適用中國國家標準（CNS ) A4規格（2l〇X 297公釐1 (請先閲讀背面之注意事項再填寫本頁) •裝· 訂· 1221482 A7 B7 五、發明説明（103) 半胱胺酸殘基195(C195)，C202，C277，C319，和C400的鼠 OPG密碼子改變成絲胺酸。然後將該muOPG基因分殖在 pcDNA 3.1(+)媒體（invitrogen，San Diego，CA)的 Not I和Xbal 兩部位之間。將所得質體，pcDNA 3.1-muOPG，及突變產生引子用Pfu聚合酶在去氧核苷酸存在中處理後，依上述在熱循環器中擴增。取一液份的反應物經由熱震盪轉染到勝任的大腸桿菌XLl_Blue中，然後予以平板培養。從轉形體所得質體DNA再經定序以查證突變。使用下列引子配對將鼠OPG基因所含半胱胺酸殘基195的密碼子改變成絲胺酸的密碼子，導到muOPG[22,401] C195S 蛋白質的產生： 1389-19 ： 5'-CAC GCA AAA GTC GGG AAT AGA TGT CAC-31 (SEQ ID NO ： 150) 1406-38 ： 5，-GTG ACA TCT ATT CCC GAC TTT TGC GTG-3, (SEQ ID NO : 151) 使用下列引子配對將鼠OPG基因所含半胱胺酸202的密碼子改變成絲胺酸的密碼子，導致muOPG[22-401] C202S蛋白質之產生：經濟部中央標準局員工消費合作社印裂 (請先閱讀背面之注意事項再填寫本頁) 1389-21 ： 5'-CAC CCT GTC GGA AGA GGC CTT CTT C-3' (SEQ ID NO ： 152) 1389-22 ： 5'-GAA GAA GGC CTC TTC CGA CAG GGT G-3f (1389-22) (SEQ ID NO : 153) -106- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 _ 五、發明説明（104) 使用下列引子配對將鼠OPG基因所含半胱胺酸殘基277的密碼子改變成絲胺酸的密碼子，導致muOPG[22-401] C277S 蛋白質之產生·· 1389-23: 5f-TGA CCT CTC GGA AAG CAG CGT GCA-3* (SEQ ID NO ： 154) 1389-24 ： 5f-TGC ACGCTGCTTTCCGAGAGGTCA-31 (SEQ ID NO ： 155) 使用下列引子配對將鼠OPG基因所含半胱胺酸殘基3 19的密碼子改成絲胺酸的密碼子，導致muOPG[22-401] C319S蛋白質之產生： 1389-17 : 5f-CCT CGA AAT CGA GCG AGC AGC TCC-3f (SEQ ID NO ： 156) 1389-18 ： 5’-CGA TTT CGA GGT CTT TCT CGT TCT C-3’（SEQ ID NO : 157) 使用下列引子配對將鼠OPG基因所含半胱胺酸殘基400的密碼子改成絲胺酸的密碼子，導致muOPG[22-401] C400S蛋白質的產生： 1406-72 : 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 5'-CCG TGA AAA TAA GCT CGT TAT AAC TAG GAA TGG-3f (SEQ ID NO : 158) 1406-75 ： 5，-CCA TTC CTA GTT ATA ACG AGC TTA TTT TCA CGG-3,（SEQ ID NO : 159)1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the Invention (100) The statement that the hydrogen sulfur group is oxidized to form a dimer is consistent. The expected mature OPG polypeptide contains 23 cysteine residues, 18 of which are predicted to participate in the formation of interchain disulfide bridges, including four cysteine-rich domains (Figure 12A). The five remaining C-terminal cysteamine residues are not involved in the secondary structure, which can be predicted based on homology with other members of the TNFR family. As a whole, there are net non-even number of photocysteine residues, and formally there may be at least one residue that is free to form an intermolecular disulfide bond between two OPG monomers. To help unravel OPG kinetics and monomer association patterns, pulse-tracking labeling studies were performed. CHO cells expressing muOPG [22-401] were metabolically labeled for 30 minutes in a serum-free medium containing 35S methionine and cysteine as described above. After this period, the medium was removed and replaced with a complete medium containing unlabeled methionine and cysteine at levels of about 2,000-fold excess of the original radioactive amino acid concentration. 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 12 hours after the addition, the culture was collected by removing the conditioning medium, and a conditioning medium and an adsorbed monolayer of lysate were prepared. The culture medium and lysate were clarified as described above, and then immunoprecipitated with anti-OPG antibody as described above. After washing the immunoprecipitates, they were released by boiling in a non-reducing SDS-PAGE buffer, and divided into two halves. In half of them, the reducing agent, Lu 氲 thiothioethanol, was added to a final concentration of 5% (v / v), while the other half was kept in a non-reducing condition. After the two groups of immunoprecipitates were analyzed by SDS-PAGE as described above, autoradiography was performed and exposed to the bottom film. The results are shown in Fig. 16. Samples analyzed by reducing SDS-PAGE are shown in the two lower panel groups. Yushen-103- This paper size is applicable to China National Standard (CNS) A4 specification (210X297mm) --------- ^-pack-(Please read the precautions on the back before filling this page) Order 1221482 A7 B7 5. After the description of the invention (101), the OPG polypeptide is rapidly processed into a slightly larger polypeptide, which may exhibit an N-linked glycosylated modified form. After about 1-2 hours, the levels in cells were sharply decreased, and simultaneously appeared in the culture supernatant. This phenomenon appears to be the result of OPG being transported from the cell to the culture medium over time, consistent with the statement that OPG is a naturally secreted protein. The analysis of the same immunoprecipitates under non-reducing conditions revealed the relationship between the formation of OPG dimers and their secretion into the conditioning medium (Figure 16, upper panel). Within the first 30-60 minutes, OPG monomers are treated in the cell by a pronounced glycosylation reaction, followed by the formation of a dimer. Over time, most of the OPG monomers are formed as dimers, which then disappear from the cells. Starting at about 60 minutes after synthesis, the 0PG dimers appeared in the conditioning medium and accumulated as the experiment continued. After this period, OPG dimers are formed, which are then secreted into the culture medium. OPG monomers are maintained in cells at low levels over time, and a small amount is also present in the culture medium. This does not seem to be the result of the covalently bonded PG dimer decomposition, but is caused by the substoichiometric amount of monomer produced in the cell and subsequently secreted. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs --------- ^-Pei-(Please read the precautions on the back before filling out this page) OPG produced from transfected CHO cells is mainly dimer Thing. To determine whether dimerization is a natural procedure in OPG synthesis, we analyzed the conditioning media of cell lines that naturally express OPG. The CTLL-2 cell line, a murine cytotoxic T lymphocyte line (ATCC accession number TIB_214), was found to express OPG mRNA in a set of tissue and cell line RNAs. The OPG transcript was the same as the 2.5-3.0 kb RNA that was identified and sequenced from the kidney and was found to encode a secretory molecule. Conditioned culture media obtained from CTLL-2 cells showed that most of them were secreted if not all -104- This paper is in accordance with Chinese National Standard (CNS) A4 (21〇X 297 mm) 1221482 Employees' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, Cooperative Printing Co., Ltd. A7 B7 V. Description of the Invention (102) PG protein is a dimer (Figure 17). At this point, it can be speculated that pG dimerization and secretion are not caused by artificial overexpression in the cell line, but may be the main product form when the product is produced by expressive cells. Normal and foreign gene-introduced mouse tissues and sera were analyzed to determine the nature of the OPG molecule exhibited in the introduction of foreign genes in OPG. Since the mouse OPG cDNA is expressed under the control of hepatocytes, extracts obtained from non-reducing conditions of control mice and mice introduced with foreign genes were used for analysis (Figure i 8). OPG dimers and stoichiometric amounts of monomers were successfully detected in extracts from mice with foreign genes introduced instead of control mice. The OPG dimers and monomers appear to be identical to the recombinant murine proteins expressed in genetically engineered CHO cells. This point strongly speculates that the OPG dimer is indeed a natural form of the gene product and may be a key active ingredient. Serum samples obtained from control mice and mice introduced with foreign genes were also similarly analyzed by Western blot analysis analysis. In the control mice, most of the OPG proteins moved in the form of dimers, and a small amount of monomers were also detected. In addition, a significant amount of a larger OPG-related protein 'was also detected, which shifted with a relative molecular weight consistent with the predicted covalently linked dimer ^. Therefore, recombinant OPG is mainly expressed as a dimer protein in OPG-introduced foreign gene mice, and the dimeric form may be the basis of the osteopetrochemical phenotype in OPG mice. OPG recombinant protein can also exist in higher molecular weight "participants" form. To determine whether the 5 C-terminal cysteine residues contained in OPG have a role in homodimerization, Use the QuickChange ™ Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA) as described above. -105-This paper size applies the Chinese National Standard (CNS) A4 specification (2l0X 297mm 1 (Please read first Note on the back, please fill in this page again) • Binding · Binding · 1221482 A7 B7 V. Description of the invention (103) The murine OPG codons of cysteine residues 195 (C195), C202, C277, C319, and C400 are changed to Serine. The muOPG gene was then cloned between Not I and Xbal in pcDNA 3.1 (+) media (invitrogen, San Diego, CA). The resulting plastid, pcDNA 3.1-muOPG, and mutations were used to generate primers. After treatment with Pfu polymerase in the presence of deoxynucleotides, it was amplified in a thermal cycler as described above. One aliquot of the reaction was transfected into competent E. coli XLl_Blue via thermal shock, and then plate cultured. The plastid DNA obtained from the transformants is then sequenced for investigation. The following primer pairing was used to change the codon of cysteine residue 195 contained in the murine OPG gene to the codon of serine, leading to muOPG [22,401] C195S protein production: 1389-19: 5'-CAC GCA AAA GTC GGG AAT AGA TGT CAC-31 (SEQ ID NO: 150) 1406-38: 5, -GTG ACA TCT ATT CCC GAC TTT TGC GTG-3, (SEQ ID NO: 151) Use the following primers to pair the mouse OPG The codon of cysteine 202 contained in the gene is changed to the codon of serine, which results in the production of muOPG [22-401] C202S protein: Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back first) Refill this page) 1389-21: 5'-CAC CCT GTC GGA AGA GGC CTT CTT C-3 '(SEQ ID NO: 152) 1389-22: 5'-GAA GAA GGC CTC TTC CGA CAG GGT G-3f ( (1389-22) (SEQ ID NO: 153) -106- This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm) 1221482 A7 B7 _ 5. Description of the invention (104) Use the following primers to pair the mouse OPG The codon of cysteine residue 277 contained in the gene is changed to the codon of serine, resulting in muOPG [22-401] C277S protein 1389-23: 5f-TGA CCT CTC GGA AAG CAG CGT GCA-3 * (SEQ ID NO: 154) 1389-24: 5f-TGC ACGCTGCTTTCCGAGAGGTCA-31 (SEQ ID NO: 155) Use the following primer pairs to pair The codon of cysteine residue 3 19 contained in the mouse OPG gene was changed to the codon of serine, resulting in the production of muOPG [22-401] C319S protein: 1389-17: 5f-CCT CGA AAT CGA GCG AGC AGC TCC-3f (SEQ ID NO: 156) 1389-18: 5'-CGA TTT CGA GGT CTT TCT CGT TCT C-3 '(SEQ ID NO: 157) The following primers were used to match the cysteine contained in the murine OPG gene The codon of residue 400 was changed to a codon of serine, which resulted in the production of muOPG [22-401] C400S protein: 1406-72: Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before (Fill in this page) 5'-CCG TGA AAA TAA GCT CGT TAT AAC TAG GAA TGG-3f (SEQ ID NO: 158) 1406-75: 5, -CCA TTC CTA GTT ATA ACG AGC TTA TTT TCA CGG-3, (SEQ ID NO: 159)

I -107- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐) ' 1221482 經濟部中央標準局員工消費合作社印製 A7 ---^__ 五、發明説明（105) 然後將含有恰當突變的每一所得mu〇PG[22-401]質體轉染到人類293細胞内，並依上述從調理培養基純化出突變〇PG- T7 — C融合蛋白質。使用實施例1 1中所述的活體外蝕骨細胞形成檢定來評估每一蛋白質的生物活性。從每一轉形體所得 "周理培養基經由用於-OPG抗體以非還原性SDS-PAGE和西方吸潰予以分析。五個C_端半光胺酸殘基中的任何一者之突變導致主要 (>90%)爲單體55 kd 0PG分子之產生。此強烈地推測該等c-端半胱胺酸殘基一起在OPG同元二體化中產生作用。構成C-端OPG缺失突變株以製作對OPG同元二體化具重要性的OPG C -端域之染色體圖區。使用可將預成熟停止轉譯信號導到鼠OPG所含C _端區内之引子經由PCR擴增構成OPG 突變株。將51寡核苷酸設計到MuOPG起始密碼子（含有 Hindlll限制部位）並將3 1寡核甞酸（含有停止密碼子和Xhol部位）設計成可將於蘇胺酸殘基200(CT 200)，脯胺酸212(CT 212)，穀胺酸293(CT-293)，或絲胺酸355(CT-355)中任一者終結的muOPG所含C-端區截頭者。使用下列引子來構成muOPG[22-200]: 1091-39 ： 5f-CCT CTG AGC TCA AGC TTC CGA GGA CCA CAA TGA ACA AG-3' (SEQ ID NO : 160) 1391-91 ·I -107- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) '1221482 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 --- ^ __ 5. Description of the invention (105) Each of the obtained muPG [22-401] plastids with appropriate mutations was transfected into human 293 cells, and the mutant OPG-T7-C fusion protein was purified from the conditioning medium as described above. The in vitro ex vivo osteoclast formation assay described in Example 11 was used to evaluate the biological activity of each protein. The " Periodic Medium " obtained from each of the transformants was analyzed via non-reducing SDS-PAGE and Western blotting for -OPG antibodies. Mutation of any of the five C-terminal semi-photogenic amino acid residues resulted in the production of a predominantly (> 90%) 55 kd 0PG molecule. This strongly speculates that these c-terminal cysteine residues together play a role in OPG homodimerization. A C-terminal OPG deletion mutant was constructed to make a chromosomal map region of the OPG C-terminal domain which is important for OPG homodimerization. OPG mutants are constructed by PCR using primers that guide the premature stop translation signal into the C-terminal region contained in the murine OPG. The 51 oligo was designed to the MuOPG start codon (containing the Hindlll restriction site) and the 3 1 oligonucleotide (containing the stop codon and Xhol site) was designed to place the threonine residue 200 (CT 200 ), Proline 212 (CT 212), glutamate 293 (CT-293), or serine 355 (CT-355) terminated muOPG contains a truncated C-terminal region. The following primers were used to construct muOPG [22-200]: 1091-39: 5f-CCT CTG AGC TCA AGC TTC CGA GGA CCA CAA TGA ACA AG-3 '(SEQ ID NO: 160) 1391-91 ·

5f-CCT CTC TCG AGT CAG GTG ACA TCT ATT CCA CAC TTT TGC -108 -5f-CCT CTC TCG AGT CAG GTG ACA TCT ATT CCA CAC TTT TGC -108-

國國家標準（CNS ) A4規格（210X297&tT (請先閲讀背面之注意事項再填寫本頁) -裝- 、?τ- 1221482 經濟部中央標準局員工消費合作社印製 A7 B7五、發明説明（106) GTGGC-3'(1391-91) (SEQ ID NO ： 161) 使用下列引子來構成muOPG[22-212]: 1091-39 ： 5f-CCT CTG AGC TCA AGC TTC CGA GGA CCA CAA TGA ACA AG-3f (SEQ ID NO ： 162) 1391-90 : 5'-CCT CTC TCG AGT CAA GGA ACA GCA AAC CTG AAG AAG GC-3’（SEQ ID NO : 163) 使用下列引子來構成muOPG[22-293] ·· 1091-39 ： 5丨-CCT CTG AGC TCA AGC TTC CGA GGA CCA CAA TGA ACA AG-3 丨 (SEQ ID NO : 164) 1391-89 : 5，-CCT CTC TCG AGT CAC TCT GTG GTG AGG TTC GAG TGG CC-3, (SEQ ID NO ： 165) 使用下列引子來構成muOPG[22-355]: 1091-39 ： 5--CCT CTG AGC TCA AGC TTC CGA GGA CCA CAA TGA ACA AG_3, (SEQ ID NO : 166) 1391-88 : 5丨-CCT CTC TCG AGT CAG GAT GTT TTC AAG TGC TTG AGG GC_3· (SEQ ID NO : 167) 然後將含有恰當截頭的每一所得muOPG_ct質體轉染到人-109- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) -裝· 、11 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（1〇7) 類293細胞内，及依上述從調理培養基純化出突變〇PG-Fc融合蛋白質。依實施例1 1中所述活體外蚀骨細胞形成檢定評估每一蛋白質的生物活性。此外也用抗-OPG抗體經由非還原性SDS-PAGE和西方吸潰來分析調理培養基。 OPG C-端區的截頭會影響OPG形成同元二體物的能力。 CT 3 55主要爲單體物，不過有某些二體物會形成。CT 293 形成顯得是等莫耳量的單體和二體，以及高分子量的凝集體。不過，CT 212和CT 200皆爲單體物。實施例1 0 OPG的绅Μ匕 Α.哺乳動物OPG-Fc融合蛋白質之純化依下述從表現OPG-Fc融合蛋白質的293細胞製備5升的調理培養基。將冷凍細胞樣品解凍到1 0毫升293S培養基中 (DMEM-高葡萄糖，lx L-葡萄糖胺，10%熱活化胎牛血清 (FBS)和100微克/毫升潮黴素）並於一天後飼與新鮮培養基。三天後，將細胞以1 : 10和1 : 20稀釋度分到兩個T175燒瓶内。另外做兩個1 : 10分流以擴大到200 T175燒瓶。細胞於此點爲後解凍5天。將細胞生長到接近會合（約三天），於此時將含血清的培養基予以攪動，用25毫升PBS每燒瓶洗細胞一次後，於每一燒瓶中加入25毫升的SF培養基（DMEM-高葡萄糖，lx L-葡萄糖胺）。將細胞保持在5% C02三天，於此點，收取培養基，予以離心，並濾過0.45m纖維素硝酸酯濾器（Corning) 0 使用在PBS中平衡過的蛋白質G Sepharose管柱（Pharmacia) -110- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） ---------裝------訂-----_線 (請先閲讀背面之注意事項再填寫本頁) 1221482 經濟部中央標準局員工消費合作社印製 A7 _B7_五、發明説明（1〇8) 純化OPG-Fc融合蛋白質。管柱尺寸係依起始培養基的體積而變異。依上述製備成的調理培養基裝載在管柱上，用PBS 洗該管柱。並手100 mM甘胺酸PH 2.7洗提純蛋白質。將洗提液份收集到裝著1M Tris pH 9.2的管子内以期儘可能快速地中和。將含蛋白質的邵份集中，於Amicon Centricon 10或 Centriprep 10中濃縮後，透遽（diafiltered)到PBS中。將純蛋白質貯存在-80°C。以這種程序純化鼠[22_401]-Fc，鼠[22_180]-Fc，鼠[22-194]-Fc，人類[22-401]-Fc和人類[22-201]Fc。鼠[22-185]-Fc 係以這種程序純化的。 B. 抗-OPG抗體之製備用muOPG[22-401]-Fc融合蛋白質皮下注射三隻紐西蘭白兔子（5·8磅初始重量）。在第一天用乳化在等體積Freunds完全佐劑中的50微克抗體將每一隻兔子免疫化。改用Freunds不完全佐劑以相同的程序進行後續加強（第1 4和2 8天）。以EIA 監測抗體效價。於第二次加強後，抗血清顯露出高抗體效價，從每一隻動物取得25毫升的生產血液。將血清先通過已固定化鼠OPG- F c的親和性管柱。用含有1 %以冰醋酸的 Pierce Gentle Elution Buffer洗提抗-OPG抗體。然後將洗提出的蛋白質透析到PBS中並通過Fc管柱以脱除對〇PG融合蛋白質的F c部份具特定性之任何抗體。將含有抗· 0PG特定性抗體的洗提分液透析到PBS中。 C. 鼠OPG丨22-4011的純化抗體親和j析術 (請先閲讀背面之注意事項再填寫本頁) •裝· 訂 -111 - 本紙張尺度適用中國國準（CNS ) A4規格(210X297公釐了 8 4 21 2 經濟部中央標準局員工消費合作社印製 A7 _B7_ 五、發明説明（109) 將經親和純化過的抗-OPG抗體透濾到偶合緩衝液（〇. 1 Μ 碳酸納 ’ pH 8.3 ’ 0· 5Μ NaCl)中’並與 CNBr_ 活化 sepharose 珠粒（Pharmacia)在室溫下混合兩小時。然後用偶合緩衝液充分地洗該樹脂後，用1M乙醇胺（pH 8.0)在室溫下將未經佔住的部位封阻住。接著用低pH洗樹脂（0.1M乙酸鈉pH 4.0，0.5M NaCl)接著以高 pH 洗（0.1M Tris-HCl pH 8.0，0.5M NaCl)。將最後的洗滌重複三次。將樹脂於最後用PBS平衡後，再填充到管柱内。一填充後，用PBS洗樹脂。用0.1M甘胺酸-HC1，pH 2·5進行對照洗提，接著用PBS再平衡。將表現muOPG[22-410]的CH0細胞所得濃縮調理培養基以低流速施加到管柱上。用PBS洗該管柱直到在280 nm測得的 11乂吸光度回歸到基線爲止。先用〇.1]^1甘胺酸-11(：1化112.5)從該管柱洗提蛋白質，用PBS再平衡，再用第二種緩衝液 (0.1M CAPS，pH 10.5，1M NaCl)洗提。將兩份洗提份分別透濾到PBA内並無菌過濾後，於-20°C下冷凍。傳統層析術將CH0細胞調理培養基置於Amicon螺旋盤繞筒（S10Y10)中濃縮23x後，透濾到20mM tris pH 8.0。然後將透濾過的培養基施加到已用20mM tris pH 8.0平衡過的Q-sepharose HP (Pharmacia)管柱。接著洗該管柱直到280 nm的吸光度達到基線爲止。用20管柱體積梯度的0-300mM NaCl/tris pH 8.0)洗提蛋白質。對管柱洗提份以西方氏吸潰偵檢OPG蛋白質。將含有OPG的洗提份合併並使其達到300mM NaCl，0.2mM DTT 的最後濃度。用 20mM tris pH 8.0，300mM NaCl， _____-112- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁) •裝· 訂 1221482 A7 B7___ 五、發明説明（110) 0.2mMDTT平衡NiNTA superose(Qiagen)管柱後，將合併洗提份施加上去。用平衡緩衝液洗該管柱到已達基線吸光度爲止。用0-30mM咪唑梯度/平衡緩衝液從管柱洗提蛋白質。用1 Μ咪唑從管柱洗出殘餘的蛋白質。再度地，用西方氏吸潰來偵檢含OPG的洗提份。將得自NiNTA管柱的合併洗提份液透析到10mM磷酸鉀pH 7.0，0.2mM DTT之中。然後將透析過的合併液施加到已平衡在1 OmM磷酸鹽緩衝液中的陶资經磷灰石管柱（Bio-Rad)。在管柱洗滌後，用20管柱體積的10_100mM磷酸鉀梯度洗提蛋白質。其後接著用20管柱體積的100-400mM磷酸鹽梯度洗提。以考馬斯藍染色SDS-聚丙晞醯胺凝膠及西方氏吸潰偵檢 OPG。將洗提份合併並透濾到PBS中，再於-80°C冷凍。純化的蛋白質係以單體形式流跑且在透濾到PBS中之後仍保持如此。於冷凍辟存或貯存在pH 5 4°C下時，該單體具安定性。不過若貯存在4°C PBS中時，於一星期後即形成二體物及似乎爲三體物和四體物者。 D.自大腸桿菌純化人類OPG met『22_4011 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 將細菌細胞糊用低切變勻化器在5 °C下懸浮在1 OmM EDTA 中到15%(w/v)之濃度。然後將細胞以兩次各在5 °C下， 15,000 psi的勻化予以破裂。將所得勻化物以5,000 X g在5°C 離心1小時。將離心丸以低切變勻化洗到水中至原有的勻化體積，接著依前述予以離心。之後，用（最後濃度）6M胍 HC1，lOmM二硫蘇糖醇，10mM Tris HC1，pH 8.5，溶液將 -113- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 48 1X 2 12 A7 B7 五、發明説明（111) (請先閱讀背面之注意事項再填寫本頁) 洗過的丸在室溫下溶解化3 0分鐘。將此溶液稀釋30-倍到含有50 mM CAPS，pH 10.5，ImM還原穀胱甘肽的2M脲中後，置於5 °C下攪拌72小時。於25°C下，先用乙酸將此溶液調整到pH 4.5，再於已用25 mM乙酸鈉，pH 4.5平衡過的SP-HP Sepharose樹脂管柱上進行層析術純化出OPG。使用2 0管柱體積的50 mM至550 mM在相同緩衝液中的線型氯化鋼梯度以0.1管柱體積/分的流速進行'管柱洗提。將只含有所欲 0PG形式的尖峯洗提份合併，貯存在5°C或經緩衝液交換到磷酸鹽緩衝食鹽水中，予以超濾濃縮，再貯存於5 °C下。以逆相HPLC，SDS-PAGE，偵檢内毒素存在性的limulus amebocyte溶裂物檢定，及N-端定序分析該物質。此外，也使用質譜測定法，pH/溫度安定性，螢光，圓偏光二色性，差式掃描熱量測定法，及蛋白酶剖面檢定諸技術來檢驗該蛋白質的折疊本質。實施例1 1 重組0PG的生物活性經濟部中央標準局員工消費合作社印製根據組織學和組織形態學，顯示出在導入外來基因小鼠内的0PG之肝中過度表現明顯地減少蝕骨細胞的數目導致骨組織的明顯增加（參看實施例4)。爲了進一步洞察在這種活體内效應底下的潛在機制，乃在蚀骨細胞形成的活體外培養模型（蝕骨細胞形成檢定）中試驗各種形式的重組OPG。這種培養系統原先係由Udagawa所構想出的（Udagawa et al. Endocrinology 125· 1805-1813(1989)，Proc. Natl· Acad· Sci· USA ϋ，7260-7264(1990))且採用骨髓細胞與來自骨髓基質 -114- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（112) 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 細胞系的細胞之組合。有關這些研究所用此培養系統的修改之説明已在以前發表過（Lacey et al. Endocrinology 136· 2367-2376(1995))。於此方法中，將從小鼠股和脛沖洗出的骨髓細胞置於增補500 U/毫升CSF-1(菌落刺激因子1，也稱爲M-CSF)- —種對單核細胞/巨噬細胞族系的細胞具特定性之造血生長因子的培養基（含有10%熱活化胎牛血清的從 MEM)中培養整個晚上。在此培育之後，收集非吸附性細胞，施以梯度純化，然後與來自骨髓細胞系ST2的細胞共培養（1 X 106非吸附性細胞：1 X 105 ST2細胞/毫升培養基）。用 ***（dexamethasone)(100 nM)和稱爲1,25-二經維生素 D3的維生素D3之生物活性代謝物（l，25(OH)2 D3，10 nM)補充該培養基。爲了增強蝕骨細胞的出現，於某些培養物中加入***素E2(250 nM)。培養期通常爲8-10天而培養基係每3 - 4天換新一次，每次將所有補充物新鮮地加入。以不同的間隔，用組織化學染色（Sigma Kit # 387A，Sigma，St· Louis，MO)或TRAP溶液檢定測定培養物的酒石酸鹽酸性磷酸酶（TRAP)之存在。該TRAP組織化學法可用來表現型地鑑定也爲TRAP+的多核（> 3核）細胞之蝕骨細胞。溶液檢定包括將食蝕骨細胞的培養物置於含0.1% Triton X-100的擰檬酸鹽緩衝液（100 mM，pH 5.0)中予以溶裂。然後根據在室溫下 3-5分鐘培育中於80 mM酒石酒鈉存在中所發生的對-硝基苯基磷酸酯（20 nM)變成對-硝基酚之轉化測量抗酒石酸性酸性磷酸酶活性。經由添加NaOH到0.5 Μ之最後濃度終止該反應。測量在405 nm的光學密度並將結果標繪出。 -115- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 _ B7___ _ 五、發明説明（113) 使用蝕骨細胞形成檢定的先前研究（Udagaws et al.同上）證明這些細胞可表現出125l·降血鈣素的受體（放射顯跡術）且可在骨表面上造成坑，其在與TRAP陽性結果組合時可確定該多核化細胞具有蝕骨細胞表現型。支持在活體外蝕骨細胞形式檢定中所得多核化細胞的蚀骨細胞表現型之其他證據爲該細胞可在免疫細胞化學中表現π v和0 3整合素 (integrins)及在原位（in situ)雜合（ISH)中表現降血鈣素受體和 TRAP mRNA。從CHO細胞調理培養基純化出huOPG[22-401]-Fc融合體並於隨後用於蝕骨細胞形成檢定中。在100毫微克/毫升 huOPG[22-401]-Fc下，蝕骨細胞形成幾乎1〇〇〇/0被抑制（圖 19A)。在微滴板洞中所裝溶裂培養物内測得之TRAP水平也會在OPG存在中被抑制，其ID50爲約3毫微克/毫升（圖20)。溶裂物中的TRAP活性水平似乎與TRAP細胞化學所看到的相對蝕骨細胞數目有相互關聯（比較圖19A-19G與20)。經純化的人類IgGl和TNFbp也在此模型中檢驗且經發現沒有抑制或刺激效應，可推測huOPG[22-401]-Fc的抑制效應係來自於融合蛋白質的OPG部份。另外也檢試人類和鼠分子的其他形式且其累積結果都摘錄於表1中。表1 各種OPG形式對活體外蝕骨細胞形成之影響 OPG構成物活體外相對生物活性 muOPG[22-401]-Fc +++ muOPG[22_194]_Fc +++ muOPG[22-185]-Fc ++ muOPG[22-180]-Fc - -116- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） ---------------IT----- (請先閱讀背面之注意事項再填寫本頁) 48 1X 2 12 A7 B7 五、發明説明（114) muOPG[22-401] +++ muOPG[22-401] C195 +++ muOPG[22-401] C202 + (請先閱讀背面之注意事項再填寫本頁) muOPG[22-401] C277 - muOPG[22-401] C319 + muOPG[22-401] C400 + muOPG[22-185] - muOPG[22-194] ++ muOPG[22-200] ++ muOPG[22-212] - muOPG[22-293] +++ muOPG[22-355] +++ huOPG[22-401]-Fc +++ huOPG[22-201]-Fc +++ huOPG[22-401]-Fc P26A +++ huOPG[22-401]-Fc Y28F +++ huOPG[22-401] +++ huOPG[27-401]-Fc ++ huOPG[29-401]-Fc ++ huOPG[32-401]-Fc +/- +++，ED5〇 = 0.4-2毫微克/毫升 ++，ED5〇 = 2_10毫微克/毫升 +，ED5〇 = 10-100毫微克/毫升 -，ED5〇 > 100毫微克/毫升經濟部中央標準局員工消費合作社印製該累積數據可推測鼠和人類OPG胺基酸序列22-401在融合到F c域，或未融合時係具有完整活體外活性者。彼等係以劑量依賴性方式抑制且具有在2-10毫微克/毫升範圍内之半-最大活性。鼠C-端在蘇酸殘基180處截頭會使分子抑活化，而在半胱胺酸185及以後者之處截頭時具有完全活性。在位置185處的半胱胺酸殘基經預測係在OPG域4的區域内形成- -117· 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印裂 A7 B7 五、發明説明（115) SS3键。於先前已證明將其他TNFR·相關蛋白質中的該殘基去除掉時會廢止其生物活性（Yan et al· J· Biol· Chem. 266? 12099-12104 (1994))。我們所發現所muOPG[22-180]_Fc爲不具活性而muOPG[22-185]_Fc爲活性者之事實與這些發現相一致。此點可推斷胺基酸殘基22-185界定一有OPG活性之區。這些發現顯示如在蚀骨細胞形成檢定中所試驗者，如同導入外來基因而表現的OPG—般，重組OPG蛋白質也會壓抑蚀骨細胞的形成。在連續暴露於OPG的培養物中進行探討 TRAP+細胞/?3 +細胞，F480+細胞的出現之時間過程實驗證明OPG會封阻TRAP+和卢3 +細胞的出現，但不會封阻F480+ 細胞。相反地，在對照培養物中於早在建立培養後的第4天即開始出現TRAP+和0 3 +細胞。而在OPG-處理過的培養物中只能發現F480+細胞且彼等似乎以與對照培養物在質地上相同的數目存在。如此，活體外OPG效應的機制似乎包括在單核細胞-巨噬細胞出現之後但在可表現TRAP或卢3整合素的細胞出現之前的蝕骨細胞分化的封阻步驟。這些發現整體地顯示OPG不干擾單核細胞·巨噬細胞前體從骨髓的一般生長和分化，反而顯示OPG會特定地封阻蚀骨細胞從單核細胞-巨噬細胞前體的選擇性分化。爲了更特定地測定出OPG在蚀骨細胞分化途徑中的何時具有抑制作用，乃採用一種活體外培養方法的變異法。此變異法載於（Lacey et al.，同上），其採用骨髓巨嗤細胞作爲蚀骨細胞前體。該蝕骨細胞前體係經由採取在CSF-1/M-CSF中培育整個晚上後的非吸附性骨髓細胞並將該細胞用1，〇〇〇-2,000 U/毫升CSF-1再培養4天而衍生得者。於稱爲生長期的 -118· 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐) ----------- (請先閱讀背面之注意事項再填寫本頁) 訂 1221482 A7 B7 五、發明説明（116) 4天培養後，取出非吸附性細胞。然後將屬於骨髓巨噬細胞的吸附性細胞在1，000-2,000 U/毫升CSF-1存在中接受各種處理長達2天之久。此二天期稱爲中間分化期。之後，將細胞層再沖洗，接著加入ST-2細胞（1X105細胞/毫升），地塞米松（100 nM)和l，25(OH)2 D3(10 nM)經過至少8天，其稱爲末端分化期。於此末端期中也可以加入試驗藥劑。於此末端期中獲得有關蝕骨細胞分化的表現型標記之資訊（Lacey et al.同上）0 將huOPG[22-401]-Fc(100毫微克/毫升）於中間分化期，末端分化期，或另外在兩分化期中加入以試驗其在此模型中對蝕骨細胞形成的影響。TRAP細胞化學和溶液兩種檢定都實施。溶液檢定的結果示於圖2 1中。當huOPG[22-401]-Fc 係加在中間期和末端期兩者或只加在末端分化期之時，其會抑制TRAP活性之出現。當其係加到中間期，然後從培養液沖洗掉，則huOPG[22-401]-Fc不會阻斷TRAP活性在培養物溶裂物中出現。細胞化學結果與溶液檢定數據相似。整體而言，這些觀察結果表示huOPG[22-401]-Fc只需要在末端分化期中存在使其施出對蝕骨細胞形成的所有壓抑效應。 B·活體内IL1-泛和ILl-yg刺激實驗經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) IL1於皮下注射到小鼠的頭頂時會系統地及局部地增加骨吸除作用（Boyce et al., Endocrinology 125· 1142-1150 (1989))。其系統性效應可經由高鈣血症程序予以評估而其局部效應可經由評估蝕骨細胞·媒介回應的相對大小予以組織學地評估。這些實驗的目的係爲了測定重組muOPG[22- -119- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 經濟部中央標準局員工消費合作社印製 B7 _五、發明説明（117) 401]_Fc在與IL1一般皮下注射到相同的頭頂區時是否會改變 IL1的局部性及/或系統性作用。 IL-1/?試驗將4星期大的雄小鼠（ICR瑞士白小鼠）分成下列諸處理組 (每組五隻小鼠）：對照組；IL1處理動物組（接受每天一次 2.5微克IL-1/?注射的小鼠）；低劑量mu〇PG[22-401]-Fc處理動物組（每天接受三次1微克mu〇PG[22-401]-Fc注射的小鼠）；低劑量muOPG[22-401]-Fc和IL1·卢；高劑量muOPG[22-401]-Fc處理動物組（每天接受三次1 〇微克mu〇PG[22-401]-Fc 注射的小鼠）；高劑量muOPG[22-401]-Fc和ILl_y5。所有小鼠都接受相同總數的活性因子注射或媒液注射（0.1%牛血清白蛋白/磷酸鹽緩衝食鹽水）。所有的動物組都在最後一次注射後的該天殺死。於第一次注射之前，第二次注射後4小時及第三次IL1注射後24小時，剛好在動物被殺之前，測量體重和血中游離鈣水平。於殺死後，取出頭頂並處理以製備石蠟切片。 IL1-從實驗將4星期大的雄小鼠（ICR瑞士白小鼠）分成下列諸處理組 (每組5隻）：對照組；ILl-從處理動物（每天接受一次5微克 IL1-從注射的小鼠）；低劑量muOPG[22-401]_Fc處理動物（每天接受一次10微克muOPG[22-401]-Fc注射的小鼠）；低劑量 muOPG[22-401]_Fc 和 IL1_ 從，（劑量如上）；高劑量muOPG [22-401]-Fc處理動物（每天接受三次10微克muOPG[22-401]-Fc注射的小鼠）；高劑量muOPG[22-401]-Fc和IL1-以。所有 -120- —- _— -— ____ 本紙張尺度適用中國國家標準（CNS ) A4規格（2l〇X297公釐） (請先閱讀背面之注意事項再填寫本頁) 裝· 訂線 1221482 A7National Standard (CNS) A4 Specification (210X297 & tT (please read the precautions on the back before filling out this page) -pack-,? Τ- 1221482 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Description ( 106) GTGGC-3 '(1391-91) (SEQ ID NO: 161) muOPG [22-212] is constructed using the following primers: 1091-39: 5f-CCT CTG AGC TCA AGC TTC CGA GGA CCA CAA TGA ACA AG- 3f (SEQ ID NO: 162) 1391-90: 5'-CCT CTC TCG AGT CAA GGA ACA GCA AAC CTG AAG AAG GC-3 '(SEQ ID NO: 163) Use the following primers to construct muOPG [22-293] · · 1091-39: 5 丨 -CCT CTG AGC TCA AGC TTC CGA GGA CCA CAA TGA ACA AG-3 丨 (SEQ ID NO: 164) 1391-89: 5, -CCT CTC TCG AGT CAC TCT GTG GTG AGG TTC GAG TGG CC-3, (SEQ ID NO: 165) Use the following primers to construct muOPG [22-355]: 1091-39: 5--CCT CTG AGC TCA AGC TTC CGA GGA CCA CAA TGA ACA AG_3, (SEQ ID NO: 166 ) 1391-88: 5 丨 -CCT CTC TCG AGT CAG GAT GTT TTC AAG TGC TTG AGG GC_3 · (SEQ ID NO: 167) Then each obtained muOPG_ct plastid containing the appropriate truncation was transfected into human-109- Paper size China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling out this page)-Equipment, 11 1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Description (1 〇7) Mutant PG-Fc fusion protein was purified from conditioned cells in 293 cells as described above. The biological activity of each protein was evaluated according to the in vitro osteoclast formation assay described in Example 11. In addition, the biological activity of each protein was also evaluated. Anti-OPG antibodies were analyzed for conditioning media via non-reducing SDS-PAGE and Western blotting. The truncation of the OPG C-terminal region can affect OPG's ability to form homodimers. CT 3 55 is predominantly monomeric, although some dimers are formed. CT 293 forms monomers and dimers that appear to be equimolar, as well as high molecular weight aggregates. However, both CT 212 and CT 200 are monomers. Example 10 Gentleman of OPG A. Purification of mammalian OPG-Fc fusion protein A 5-liter conditioning medium was prepared from 293 cells expressing OPG-Fc fusion protein as follows. The frozen cell samples were thawed into 10 ml of 293S medium (DMEM-high glucose, lx L-glucosamine, 10% heat-activated fetal calf serum (FBS) and 100 μg / ml hygromycin) and fed with fresh one day later Culture medium. After three days, the cells were divided into two T175 flasks at 1:10 and 1:20 dilutions. Make two additional 1:10 splits to expand to a 200 T175 flask. Cells were thawed for 5 days at this point. The cells were grown to near confluence (about three days). At this time, the serum-containing medium was agitated. After washing the cells with 25 ml of PBS per flask, 25 ml of SF medium (DMEM-high glucose) was added to each flask. , Lx L-glucosamine). The cells were kept at 5% C02 for three days. At this point, the medium was collected, centrifuged, and filtered through a 0.45m cellulose nitrate filter (Corning). 0 A protein G Sepharose column (Pharmacia) -110 equilibrated in PBS was used. -This paper size applies to China National Standard (CNS) A4 specification (210X 297 mm) --------- install ------ order -----_ line (please read the note on the back first) Please fill in this page again for details) 1221482 A7 _B7_ printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (108) Purified OPG-Fc fusion protein. The column size varies depending on the volume of the starting medium. The conditioning medium prepared as described above was loaded on a column, and the column was washed with PBS. Purify the protein by hand with 100 mM glycine pH 2.7. The eluate was collected into a tube containing 1M Tris pH 9.2 in order to neutralize it as quickly as possible. The protein-containing portion was concentrated, concentrated in Amicon Centricon 10 or Centriprep 10, and then diafiltered into PBS. Store pure protein at -80 ° C. Murine [22_401] -Fc, murine [22_180] -Fc, murine [22-194] -Fc, human [22-401] -Fc, and human [22-201] Fc were purified in this procedure. Murine [22-185] -Fc was purified by this procedure. B. Preparation of anti-OPG antibodies Three musk New Zealand rabbits (5 · 8 pounds initial weight) were injected subcutaneously with muOPG [22-401] -Fc fusion protein. Each rabbit was immunized on the first day with 50 micrograms of antibody emulsified in an equal volume of Freunds complete adjuvant. Follow the same procedure with Freunds incomplete adjuvant for subsequent enhancements (days 14 and 28). Monitor antibody titers with EIA. After the second boost, the antisera showed high antibody titers, and 25 ml of production blood was obtained from each animal. The sera were first passed through the affinity column of the immobilized murine OPG-Fc. Anti-OPG antibodies were eluted with Pierce Gentle Elution Buffer containing 1% glacial acetic acid. The eluted protein was then dialyzed into PBS and passed through an Fc column to remove any antibodies specific for the F c portion of the OPG fusion protein. The eluate containing the anti-OPG specific antibody was dialyzed into PBS. C. Mouse OPG 丨 22-4011 purified antibody affinity analysis (please read the precautions on the back before filling this page) • Binding · Order -111-This paper size applies to China National Standard (CNS) A4 specification (210X297) 8 4 21 2 A7 _B7_ printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (109) Affinity-purified anti-OPG antibody was diafiltered into a coupling buffer (0.1 M sodium carbonate 'pH 8.3 '0.5M NaCl) and mixed with CNBr_ activated sepharose beads (Pharmacia) at room temperature for two hours. Then the resin was sufficiently washed with coupling buffer, and then 1M ethanolamine (pH 8.0) at room temperature The unoccupied area was blocked. Then the resin was washed with low pH (0.1M sodium acetate pH 4.0, 0.5M NaCl) followed by high pH (0.1M Tris-HCl pH 8.0, 0.5M NaCl). The washing was repeated three times. After the resin was finally equilibrated with PBS, it was filled into the column. After filling, the resin was washed with PBS. The control was eluted with 0.1M glycine-HC1, pH 2.5. PBS rebalance. Concentrated conditioning medium obtained from CH0 cells expressing muOPG [22-410] at low flow rate Add to the column. Wash the column with PBS until the 11 乂 absorbance measured at 280 nm returns to the baseline. First remove 0.1% glycine-11 (: 112.5) from the column. The protein was eluted, re-equilibrated with PBS, and then eluted with a second buffer solution (0.1M CAPS, pH 10.5, 1M NaCl). The two eluted fractions were diafiltered into PBA and sterile filtered. Frozen at ° C. Traditionally, CH0 cell conditioning medium was placed in Amicon spiral coil (S10Y10) and concentrated 23x, and then diafiltered to 20mM tris pH 8.0. Then the diafiltration medium was applied to 20mM tris pH 8.0. An equilibrated Q-sepharose HP (Pharmacia) column. The column was then washed until the absorbance at 280 nm reached the baseline. The protein was eluted with a 20-column volume gradient of 0-300 mM NaCl / tris pH 8.0). The OPG protein was detected by Western blotting on the column elution fraction. The fractions containing OPG were combined and brought to a final concentration of 300 mM NaCl, 0.2 mM DTT. Use 20mM tris pH 8.0, 300mM NaCl, _____- 112- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the precautions on the back before filling this page) • Binding and ordering 1221482 A7 B7___ 5. Description of the invention (110) After equilibrating the NiNTA superose (Qiagen) column with 0.2mMDTT, apply the combined eluate. Wash the column with equilibration buffer until the baseline absorbance has been reached. Protein was eluted from the column with a 0-30 mM imidazole gradient / equilibrium buffer. The residual protein was washed from the column with 1 M imidazole. Once again, Western blots were used to detect OPG-containing eluted fractions. The combined eluate obtained from the NiNTA column was dialyzed into 10 mM potassium phosphate pH 7.0, 0.2 mM DTT. The dialyzed pooled solution was then applied to a ceramic apatite column (Bio-Rad) which had been equilibrated in 10 mM phosphate buffer. After column washing, the protein was eluted with a 20-column volume of a 10-100 mM potassium phosphate gradient. This was followed by elution with a gradient of 100-400 mM phosphate in a 20-column volume. Coomassie blue-stained SDS-polypropylamine gel and Western blotting were used to detect OPG. The fractions were combined and diafiltered into PBS and frozen at -80 ° C. The purified protein was run as a monomer and remained the same after diafiltration into PBS. The monomer is stable when stored frozen or stored at pH 5 4 ° C. However, if stored in 4 ° C PBS, two bodies and three bodies and four bodies appear to form after one week. D. Purification of human OPG from E. coli "22_4011 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). Bacterial cell paste with low shear homogenizer at 5 ° C Suspended in 1 OmM EDTA to a concentration of 15% (w / v). The cells were then lysed by homogenization at 15,000 psi at 5 ° C twice. The resulting homogenate was centrifuged at 5,000 X g for 1 hour at 5 ° C. The centrifugal pellets were homogenized with low shear into water to the original homogenization volume, and then centrifuged as described above. After that, use (final concentration) 6M guanidine HC1, 10 mM dithiothreitol, 10 mM Tris HC1, pH 8.5, and the solution will be -113. This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) 48 1X 2 12 A7 B7 V. Description of the invention (111) (Please read the precautions on the back before filling in this page) The washed pills dissolve at room temperature for 30 minutes. This solution was diluted 30-fold into 2M urea containing 50 mM CAPS, pH 10.5, ImM reduced glutathione, and then stirred at 5 ° C for 72 hours. At 25 ° C, the solution was adjusted to pH 4.5 with acetic acid, and then OPG was purified by chromatography on a SP-HP Sepharose resin column equilibrated with 25 mM sodium acetate, pH 4.5. A 'column elution' was performed using a 20 Column volume of 50 mM to 550 mM linear steel chloride gradient in the same buffer at a flow rate of 0.1 column volume / minute. The spiked fractions containing only the desired form of OPG were combined and stored at 5 ° C or buffer exchanged into phosphate-buffered saline, concentrated by ultrafiltration, and stored at 5 ° C. The substance was analyzed by reverse-phase HPLC, SDS-PAGE, limulus amebocyte lysate assay for detection of endotoxin presence, and N-terminal sequencing. In addition, mass spectrometry, pH / temperature stability, fluorescence, circular polarization dichroism, differential scanning calorimetry, and protease profiling techniques were also used to examine the folding nature of the protein. Example 1 1 Recombinant bioactive activity of OPG Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Based on histology and histomorphology, it was shown that the overexpression of OPG in liver introduced into mice with foreign genes significantly reduced the number of osteoclasts The number results in a significant increase in bone tissue (see Example 4). To gain further insight into the underlying mechanisms underlying this in vivo effect, various forms of recombinant OPG were tested in an in vitro culture model of osteoclast formation (osteoclast formation assay). This culture system was originally conceived by Udagawa (Udagawa et al. Endocrinology 125 · 1805-1813 (1989), Proc. Natl · Acad · Sci · USAϋ, 7260-7264 (1990)) and used bone marrow cells and From the bone marrow matrix-114- This paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 V. Description of the invention (112) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the note on the back first) (Please fill in this page for more details.) Instructions for the modification of this culture system by these institutes have been previously published (Lacey et al. Endocrinology 136 2367-2376 (1995)). In this method, bone marrow cells washed from mouse thighs and tibia are placed in supplemented with 500 U / ml CSF-1 (colony stimulating factor 1, also known as M-CSF)-a pair of monocytes / macrophages Cells of the pedigree were cultured in a specific hematopoietic growth factor medium (from MEM containing 10% heat-activated fetal bovine serum) throughout the night. After this incubation, non-adsorbent cells were collected, subjected to gradient purification, and then co-cultured with cells from the bone marrow cell line ST2 (1 X 106 non-adsorbent cells: 1 X 105 ST2 cells / ml medium). The medium was supplemented with dexamethasone (100 nM) and a bioactive metabolite of vitamin D3 called 1,25-divitamin D3 (l, 25 (OH) 2 D3, 10 nM). To enhance the appearance of osteoclasts, prostaglandin E2 (250 nM) was added to some cultures. The culture period is usually 8-10 days and the culture medium is renewed every 3-4 days, and all supplements are added fresh each time. At different intervals, the presence of tartrate acid phosphatase (TRAP) in the cultures was determined by histochemical staining (Sigma Kit # 387A, Sigma, St. Louis, MO) or TRAP solution assay. This TRAP histochemical method can be used to phenotypically identify osteoclasts of multinucleated (> 3-nucleus) cells that are also TRAP +. The assay involves lysing the culture of osteotrophic cells in 0.1% Triton X-100 citrate buffer (100 mM, pH 5.0). Anti-tartaric acidic phosphoric acid was then measured based on the conversion of p-nitrophenyl phosphate (20 nM) into p-nitrophenol in the presence of 80 mM sodium tartrate in 3-5 minutes incubation at room temperature Enzyme activity. The reaction was stopped by adding NaOH to a final concentration of 0.5 M. Measure the optical density at 405 nm and plot the results. -115- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) 1221482 Printed by A7 _ B7___ _ of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (113) Studies (Udagaws et al. Ibid.) Have demonstrated that these cells can exhibit 125l · calcitonin receptors (radiography) and cause pits on the bone surface, which can be identified in combination with TRAP-positive results Cells have the osteoclast phenotype. Other evidence supporting the osteoclast phenotype of the multinucleated cells obtained in the in vitro osteolysate cell assay is that the cell can express π v and 0 3 integrins in immunocytochemistry and in situ ) Hybridization (ISH) shows calcitonin receptor and TRAP mRNA. The huOPG [22-401] -Fc fusion was purified from CHO cell conditioning media and used in subsequent osteoclast formation assays. At 100 ng / ml huOPG [22-401] -Fc, osteoclast formation was inhibited by almost 1000/0 (Figure 19A). The TRAP levels measured in the lysed cultures contained in the microtiter plate holes were also suppressed in the presence of OPG, with an ID50 of approximately 3 ng / ml (Figure 20). The level of TRAP activity in the lysate seems to correlate with the relative number of osteoclasts seen in TRAP cytochemistry (compare Figures 19A-19G and 20). Purified human IgG1 and TNFbp were also tested in this model and were found to have no inhibitory or stimulating effect. It can be speculated that the inhibitory effect of huOPG [22-401] -Fc is derived from the OPG portion of the fusion protein. Other forms of human and murine molecules were also tested and their cumulative results are summarized in Table 1. Table 1 Effects of various OPG forms on in vitro osteoclast formation. Relative biological activity of OPG constructs in vitro muOPG [22-401] -Fc +++ muOPG [22_194] _Fc +++ muOPG [22-185] -Fc + + muOPG [22-180] -Fc--116- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) --------------- IT --- -(Please read the notes on the back before filling this page) 48 1X 2 12 A7 B7 V. Description of the invention (114) muOPG [22-401] +++ muOPG [22-401] C195 +++ muOPG [22 -401] C202 + (Please read the notes on the back before filling this page) muOPG [22-401] C277-muOPG [22-401] C319 + muOPG [22-401] C400 + muOPG [22-185]-muOPG [22-194] ++ muOPG [22-200] ++ muOPG [22-212]-muOPG [22-293] +++ muOPG [22-355] +++ huOPG [22-401] -Fc ++ + huOPG [22-201] -Fc +++ huOPG [22-401] -Fc P26A +++ huOPG [22-401] -Fc Y28F +++ huOPG [22-401] +++ huOPG [27-401 ] -Fc ++ huOPG [29-401] -Fc ++ huOPG [32-401] -Fc +/- +++, ED50 = 0.4-2 ng / ml ++, ED50 = 2_10 ng / Ml +, ED50 = 10-100 ng / ml-, ED50 > 100 ng / ml Bureau employees consumer cooperatives print the accumulated data can speculate mouse and human OPG amino acid sequence 22-401 fused to the F c domain, the Department has a complete in vitro activity by time or unfused. They are inhibited in a dose-dependent manner and have a half-maximum activity in the range of 2-10 ng / ml. The truncation of the murine C-terminus at threonine residue 180 inhibits the molecule from being activated, and is fully active when truncated at cysteine 185 and the latter. The cysteine residue at position 185 is predicted to form in the area of OPG domain 4--117 · This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 Central Bureau of Standards, Ministry of Economic Affairs Employee consumer cooperative prints A7 B7 V. Description of invention (115) SS3 key. Removal of this residue in other TNFR · related proteins has previously been shown to abolish its biological activity (Yan et al. J. Biol. Chem. 266? 12099-12104 (1994)). The fact that we found that muOPG [22-180] _Fc is inactive and that muOPG [22-185] _Fc is active is consistent with these findings. It can be inferred that amino acid residues 22-185 define a region having OPG activity. These findings show that, as tested by osteoclast formation assays, recombinant OPG proteins, like OPG expressed by the introduction of foreign genes, also suppress the formation of osteoclasts. In the continuous exposure to OPG culture, the time course of the appearance of TRAP + cells /? 3+ cells and F480 + cells was verified. OPG blocked the appearance of TRAP + and Lu3 + cells, but not F480 + cells. In contrast, TRAP + and 0 3 + cells began to appear in the control culture as early as 4 days after the establishment of the culture. Whereas only F480 + cells were found in OPG-treated cultures and they appeared to be present in the same number of textures as the control cultures. Thus, the mechanism of the OPG effect in vitro seems to include a blocking step of osteoclast differentiation after the appearance of monocyte-macrophages but before the emergence of cells that can express TRAP or Lu3 integrin. These findings collectively show that OPG does not interfere with the general growth and differentiation of monocytes and macrophage precursors from the bone marrow, but instead shows that OPG specifically blocks the selective differentiation of osteocytes from monocyte-macrophage precursors . In order to more specifically determine when OPG has an inhibitory effect in the osteoclast differentiation pathway, a mutation method using an in vitro culture method is used. This variation is described in (Lacey et al., Supra), which uses bone marrow giant cymbal cells as precursors to osteoclasts. The pre-osteolysed cell system was obtained by taking non-adhesive bone marrow cells cultured in CSF-1 / M-CSF overnight, and culturing the cells with 1,000-2,000 U / ml CSF-1 for 4 days. Derivatives. In the term of growth term -118 · This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) ----------- (Please read the precautions on the back before filling this page) Order 1221482 A7 B7 V. Description of the invention (116) After 4 days of culture, remove non-adsorbent cells. Adherent cells belonging to bone marrow macrophages were then subjected to various treatments in the presence of 1,000-2,000 U / ml CSF-1 for up to 2 days. This two-day period is called the intermediate differentiation period. After that, the cell layer was washed again, followed by the addition of ST-2 cells (1X105 cells / ml), dexamethasone (100 nM) and 1,25 (OH) 2 D3 (10 nM) for at least 8 days, which is called the end Differentiation period. Test agents can also be added during this terminal phase. Obtain information on phenotypic markers of osteoclast differentiation during this terminal phase (Lacey et al. Ibid.) 0 Put huOPG [22-401] -Fc (100 ng / ml) in the middle differentiation phase, terminal differentiation phase, or It was also added during the two differentiation stages to test its effect on osteoclast formation in this model. Both TRAP cytochemistry and solution tests are performed. The results of the solution verification are shown in FIG. 21. When huOPG [22-401] -Fc is added to both the intermediate and terminal stages or only to the terminal differentiation stage, it inhibits the appearance of TRAP activity. When it was added to the intermediate phase and then washed away from the culture medium, huOPG [22-401] -Fc did not block TRAP activity from appearing in the culture lysate. The cytochemical results were similar to the solution assay data. Taken as a whole, these observations indicate that huOPG [22-401] -Fc only needs to be present during terminal differentiation to allow it to exert all repressive effects on osteoclast formation. B. In vivo IL1-Pan and ILl-yg stimulation experiments printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) IL1 will be systematically injected subcutaneously into the mouse's head Locally increased bone resorption (Boyce et al., Endocrinology 125 1142-1150 (1989)). Its systemic effects can be evaluated by the hypercalcemia procedure and its local effects can be evaluated histologically by evaluating the relative size of the osteoclasts and mediator responses. The purpose of these experiments is to determine the recombined muOPG [22- -119- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs B7 _V. Description of the invention (117) 401] Fc will change the local and / or systemic effects of IL1 when injected subcutaneously into the same cephalic region as IL1. The IL-1 /? Test divided 4-week-old male mice (ICR Swiss white mice) into the following treatment groups (five mice per group): a control group; an IL1-treated animal group (receiving 2.5 micrograms of IL- once a day) 1 /? Injected mice); low-dose muOPG [22-401] -Fc treated animal group (mice receiving 1 μg muOPG [22-401] -Fc injected three times a day); low-dose muOPG [ 22-401] -Fc and IL1 · Lu; high-dose muOPG [22-401] -Fc-treated animals (mouse receiving 10 μg muOPG [22-401] -Fc injections three times a day); high-dose muOPG [22-401]-Fc and IL1_y5. All mice received the same total number of active factor injections or vehicle injections (0.1% bovine serum albumin / phosphate buffered saline). All animal groups were killed that day after the last injection. Prior to the first injection, 4 hours after the second injection and 24 hours after the third IL1 injection, body weight and blood free calcium levels were measured just before the animals were killed. After killing, the top of the head was removed and processed to prepare paraffin sections. IL1- From experiment, 4-week-old male mice (ICR Swiss white mice) were divided into the following treatment groups (5 in each group): control group; IL1-from treatment animals (receiving 5 micrograms of IL1-from injection once a day) Mice); low-dose muOPG [22-401] _Fc treated animals (mice receiving 10 μg muOPG [22-401] -Fc injections once a day); low-dose muOPG [22-401] _Fc and IL1_ As above); high-dose muOPG [22-401] -Fc treated animals (mouses receiving 10 μg muOPG [22-401] -Fc injections three times a day); high-dose muOPG [22-401] -Fc and IL1- to. All -120- —- _— -— ____ This paper size is applicable to Chinese National Standard (CNS) A4 (2l0X297mm) (Please read the precautions on the back before filling this page) Binding 1221482 A7

經濟部中央標準局員工消費合作社印製 =接受每天相同數目的活性因子注射或媒液注射。所有動物組都在最後一次注射後當天殺死。在第一次注^ 前’第二次注射後四小時和第三次IL1注射後24小時，剛好在動物被殺死之前’測量血液游離鈣水平。在第一次之前，第二次注射後四小時及第三次⑸注射後Μ小時，剛好在殺死動物之前，測f動物的體ΐ。在殺死後，取出頭頂並進行石蠟切片處理。組織學方法將頭頂骨固定在鋅福馬林中，於甲酸中去鈣，以乙醇脱水並安置在石蠟中。切片（5微米厚）係從鄰接人字缝的頭頂切出並用蘇木素和曙紅予以染色或針對抗酒石酸鹽性酸性磷酸酶活性進行反應（Sigma Kit # 387Α)及用蘇木素予以逆染色。經由用〇steomeasure(Osteometrics，Atlanta, GA)以安裝在顯微鏡上的攝影機明亮接著將組織學特徵示跡在數字化器的滾筒上之組織學方法評估在IL1_從處理小鼠中的骨吸除作用。測定頭頂骨的骨髓空間内的蝕骨細胞數，蝕骨細胞襯底表面，和蝕化表面。頭頂的注射側和無注射側係分開測量者。結果 ILL· α和IL1-/?會在所用劑量下，特別是第二天產生高鈣血症，可能是經由謗導出系統性增加的骨吸除所致。於IL1-卢處理小鼠中高鈣血症回應會被mu〇PG[22-401]-Fc所阻斷且在IL1-仪處理小鼠中明顯地減低，一種在第2天最明顯吟效應（圖 22A-22B)。圖IL1-以和IL1_卢處理過的小鼠之頭頂組織學分析顯示單 121 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） ------IT-----_線 (請先聞讀背面之注意事項再填寫本頁) 1221482 經濟部中央標準局員工消費合作社印製蝕骨細胞表面佔骨表面的％(平均値土80) 蝕穿表面佔骨表面的 %(平均値土 SD) 蝕骨細胞數/平方毫米組織面積(平均値土 SD) 實驗1 無注射侧注射側無注射侧注射侧無注射侧注射側對照組 12.36 土 3.44 9.54 土 2.46 8.07 土 3.90 9.75 土 3.16 32.51 土 11.09 23.50土 10.83 ILl-^(2.5//g/d) 17.18 土 130 16.0 土 21.6 40.66土 428 37.53 土 1028 Ή.80 土 18.76 60.89土 5.16 OPG(40// g/d) 10.12 土 371 5.04 土 1.66 9.73 土 4.33 4.19±3.61 32.73 土 11.09 1524 土 7.54 OPG+ILl-yS 18.61 土 2.46 #1326土 2.50 44.87 土 8.63 #25.94 土 6.82 69.42土 3629 #47.13 土 2426 實驗2 對照組 11.56 土422 11.95 土 2.97 12.67 土 5.04 10.03 土 5.13 5172 土 23.93 56.03 土3070 ILl-a(5//g/d) 28.81 ±4.84 23.46土 5.76 37.51 土 5.16 41.10 土 12.53 113.60± 18.04 102.70土 32.09 OPG(40//^d) 14.40 土 1.00 R26 土 2.54 11.55 土 4.14 #429 土 3.16 7228 土 14.11 #22·65 土 16.68 OPG+ILl-α 29.58土 8.80 #17.83 土 3.34 33.66土 921 #24·38±8·88 146.10 土 42.37 #66.56±15.62 A7 ____B7__ 五、發明説明（119) 用IL1處理會使諸種骨吸除指數明顯增加：蝕骨細胞數，姓骨細胞襯底表面；和蚀穿表面（表面因蚀骨作用而顯示出深扇形）（圖23B，表2)。對於IL1_從和ILl_y3的回應而言，骨吸除作用的增加在頭頂的注射侧和無注射側面上都類似。 MuOPG[22-401] Fc注射會減少ILl-α和卢處理過的小鼠及只接受媒液注射的小鼠之骨吸除作用，但此減少效用只出現於頭頂經muOPG[22_401]-Fc注射過的側邊。這些觀察結果的最可能解釋爲mu〇PG[22-401 ]-Fc會抑制骨吸除作用’爲可由總蚀骨細胞數和正進行骨吸除的可得骨表面之百分比兩者在鄰接mu〇PG[22-401]-Fc注射部位的區域内同時減低之事實所支持的結論。從組織學來看， muOPG[22-401]_Fc的作用係局部地最顯著，但muOPG[22- 401]-Fc也使IL1-謗發高鈣血鈣受挫之事實也可推斷出 muOPG[22-401]-Fc對骨吸除作用具有系統地更微妙之影響。表2· OPG對IL-1注射小鼠體内的骨吸除變數之影響 #相對於無注射的差異p<〇.〇5(經由配對t試驗） -122- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐j I 11 裝— 111 I 訂線 (請先閱讀背面之注意事項再填寫本頁) 1221482 經濟部中央標準局員工消費合作社印製 A 7 ____ B7 五、發明説明（120 ) C，muOPG丨22-401]-Fc在生甚中的小鼠體内之系統性影響將3-4星期大，重量9.2-15.7克的雄BDF1小鼠分成每組十隻的數組。用食鹽水或muOPG[22-401]-Fc 2·5亳克/公斤每曰兩次地皮下注射這些小鼠14天（5毫克/公斤/天）。在處理前，第7天和第14天將這些小鼠射線攝影。在最後注射24 小時後將小鼠殺死。取出右股，固定在鋅馬福林中，於甲酸内除#5並包埋在石蠛中。從遠股幹瓶端的中區和股轴切出切片。於從生長板的幹骺端限延展經過原發和續發骨鬆質到股幹（軸）的六個相鄰區以組織形態學測定法測定骨密度。每一區皆爲0.5X0.5平方毫米。射線攝影變化於處理七天後，在OPG處理小鼠體内相對於對照組已可看到與生長板缔合的骨鬆質内出現骨密度增加的區之跡象。該效果在遠股和近脛中期中特別突出（圖24Α-24Β)。不過，在椎骨體，髂螫和遠脛中也出現密度增加的帶。於第14天時，不透光區已更延展到股幹和脛幹中，雖則放射不透性的強度已減弱。此外，在實驗完成時股的長度沒有不同或在實驗進行中的長度變化也沒有不同，意謂著〇PG不會變更骨生長。組織學變化遠股幹骺端顯示出在距生長板1.1至2.65毫米區内有增加的骨密度（圖2 5和26Α-26Β)。此爲骨被小鼠内蝕骨細胞媒介骨吸除所迅速脱除掉之區。於這些快速生長中的幼鼠體内，所觀察到的在此區内用OPG處理所致骨增加現象係與骨 — ______-123- 本紙張尺度適用中國國家標準（CNS ) A*規格（21〇><297公釐） (請先閱讀背面之注意事項再填寫本頁) -絮· 、ν" 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（121) 吸除作用的抑制相一致的。 D.促骨堆積肽對於老鼠卵巢切除所謗發的骨質流失之影響將十二週大的雌費施爾鼠（Fisher rats)施以卵巢切除術 (OVX)或假手術並對遠股幹骺端的骨密度進行雙X射線吸收測定法（DEXA)測量。於三天恢復期後，於14天期間，該等動物接受下述的每天注射：十隻假手術動物接受媒液注射 (磷酸鹽緩衝食鹽水）；六隻OVX動物接受OPG-Fc 5毫克/公斤皮下注射；六隻OVX動物接受***（ESTR)40微克/公斤皮下注射。在處理七天和1 4天後，以DEXA測量動物的骨密度。在最後注射後兩天，殺死動物並切除右脛和合股供組織學評估用。骨密度DEXA測量顯示出卵巢切除後被OPG-Fc阻斷的骨質密度減少傾向。其效應類似於已知的抗吸除劑***和 pamidronate(圖2 7 )。組織形態測定法分析確定用OPG-F c處理所得這些觀察結果，產生在OVX老鼠中比在未處理的 0 VX老鼠中明顯較高的骨密度（圖2 8 )。這些結果確立OPG在與卵巢切除後的内源***退去相關的骨質流失中之活性。活體内概述重組OPG的活體内作用類似於OPG導入外來基因小鼠體内所看到的變化。OPG導入外來基因小鼠體内所看到的蝕骨細胞數目之減少可經由在正常小鼠和用IL1_從或IL1-卢處理過的小鼠之頭頂局部注射重組OPG而複現。OPG導入外來基因小鼠會發展出骨質石化性表現型，其骨髓腔内會漸進地填充入骨質且在出生後第1天起從生長板會延展出未再塑過的 -124- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ---------裝------訂-----^線 (請先閱讀背面之注意事項再填寫本頁) 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（122) 軟骨。對於正常三週大（生長中）的小鼠，OPG處理也會導致軟骨内骨形成區内的骨和未再塑軟骨之保留，一種經由射線攝影術所觀察到且由組織學確定過之效應。因此，重組 OPG會在正常動物產生類似於在導入外來基因動物體内看到的表現型之變化且該變化與OPG·謗導出的骨吸除作用之抑制一致。根據活體外蚀骨細胞形成檢定，此抑制作用的一明顯部份係受損的蝕骨細胞形成作用所致。與此假設一致者，OPG會阻斷卵巢切除術謗導的老鼠骨疏鬆病。此模型中的骨質流失係已知爲經活化的蚀骨細胞所促成者，由此暗示OPG在原發性骨疏鬆病治療中的作用。實施例1 2 OPG之聚乙二醇化衍生物經由還原性烷基化製備N-端PEG-OPG拼合物將 HuOPG met[22-194] P25A 緩衝交換到 25-50 mM NaOAc，pH 4.5-4.8内並濃縮到2-5毫克/毫升。此溶液即用來於5-7°C下以單官能性PEG醛進行OPG還原性烷基化。將線型或分枝的MW=1至5 7 kDa之PEG單官能醛（可得自 Shearwater Polymers)以固體形式可構成2-4莫耳PEG膝/莫耳 OPG之量加到該OPG溶液内。在聚合物溶解到蛋白質溶液中之後，用1-1.6 Μ新製備的在冷去離子水中的儲液將氰硼氫化鈉加到反應混合物内至15至20 mM之最後濃度。經由在 G3000SWXL 管柱（Toso Haas)上用 100 mM NaP04，0.5 Μ NaCl，10°/〇乙醇，pH 6.9洗提進行尺寸排斥HPLC(size exclusion HPLC)以監測反應的進展和OPG聚乙二醇化的程 -125- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） ----------- (請先閱讀背面之注意事項再填寫本頁)Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs = Receive the same number of injections of active factors or vehicle injections per day. All animal groups were killed the day after the last injection. Blood free calcium levels were measured before the first injection, 'four hours after the second injection and 24 hours after the third IL1 injection, just before the animal was killed'. Before the first injection, four hours after the second injection, and M hours after the third injection, the body size of the animal was measured just before the animal was killed. After killing, the head was removed and paraffin sectioned. Histological method The parietal bone was fixed in zinc formalin, decalcified in formic acid, dehydrated with ethanol and placed in paraffin. Sections (5 microns thick) were excised from the top of the head adjacent to the herringbone slit and stained with hematoxylin and eosin or reacted with tartrate-resistant acid phosphatase activity (Sigma Kit # 387A) and counterstained with hematoxylin. Evaluation of IL1_ bone resorption from treated mice by histological method using osteomeasure (Osteometrics, Atlanta, GA) with a camera mounted on a microscope followed by histological features on a digitizer roller . The number of osteoclasts in the bone marrow space of the parietal bone, the osteoclast substrate surface, and the eroded surface were measured. The injection side and the non-injection side of the head are separated from the measurer. Results ILL · α and IL1- /? Will produce hypercalcemia at the dosage used, especially the next day, which may be caused by systemic increase of bone resorption through defamation. Hypercalcemia response in IL1-Lu-treated mice was blocked by muOPG [22-401] -Fc and was significantly reduced in IL1-Meter-treated mice, one of the most pronounced chanting effects on day 2 22A-22B). Figure IL1- Histological analysis of the head of mice treated with and IL1_Lu show single 121-This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) ------ IT --- --_ line (please read the precautions on the back before filling out this page) 1221482 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, the surface of osteoclasts occupies the bone surface as a percentage of the average bone surface (average soil 80). % (Mean soil SD) bone erosion cells / mm2 tissue area (mean soil SD) Experiment 1 No injection side injection side No injection side injection side No injection side injection side Control group 12.36 3.43.44 9.54 2.42.46 8.07 土3.90 9.75 soil 3.16 32.51 soil 11.09 23.50 soil 10.83 ILl-^ (2.5 // g / d) 17.18 soil 130 16.0 soil 21.6 40.66 soil 428 37.53 soil 1028 Ή.80 soil 18.76 60.89 soil 5.16 OPG (40 // g / d) 10.12 soil 371 5.04 soil 1.66 9.73 soil 4.33 4.19 ± 3.61 32.73 soil 11.09 1524 soil 7.54 OPG + ILl-yS 18.61 soil 2.46 # 1326 soil 2.50 44.87 soil 8.63 # 25.94 soil 6.82 69.42 soil 3629 # 47.13 soil 2426 Experiment 2 Control group 11.56 soil 422 11.95 soil 2.97 12.67 soil 5.04 10.03 soil 5. 13 5172 soil 23.93 56.03 soil 3070 ILl-a (5 // g / d) 28.81 ± 4.84 23.46 soil 5.76 37.51 soil 5.16 41.10 soil 12.53 113.60 ± 18.04 102.70 soil 32.09 OPG (40 // ^ d) 14.40 soil 1.00 R26 soil 2.54 11.55 soil 4.14 # 429 soil 3.16 7228 soil 14.11 # 22 · 65 soil 16.68 OPG + ILl-α 29.58 soil 8.80 # 17.83 soil 3.34 33.66 soil 921 # 24 · 38 ± 8 · 88 146.10 soil 42.37 # 66.56 ± 15.62 A7 ____B7__ 5. Description of the invention (119) Treatment with IL1 significantly increases the bone resorption index: the number of osteoclasts, the surficial surface of the osteocytes, and the erosive surface (the surface shows a deep fan shape due to osteogenesis) (Figure 23B, Table 2). For IL1_slave and ILl_y3's response, the increase in bone aspiration was similar on the injection side and the non-injection side of the head. MuOPG [22-401] Fc injection reduced the bone resorption effect of IL1-α and Lu-treated mice and mice that received only vehicle injection, but this reduction effect appeared only at the top of the head via muOPG [22_401] -Fc Injected side. The most likely explanation for these observations is that muOPG [22-401] -Fc inhibits bone resorption. 'It is the total number of osteoblasts that can be etched and the percentage of available bone surface that is undergoing bone resorption. The conclusion supported by the fact that the area of the PG [22-401] -Fc injection site is simultaneously reduced. From a histological point of view, the role of muOPG [22-401] _Fc is locally the most significant, but the fact that muOPG [22-401] -Fc also frustrated IL1-defeated hypercalcemia can also be inferred that muOPG [22 -401] -Fc has a more subtle effect on bone resorption. Table 2. The effect of OPG on bone resorption variables in IL-1 injected mice #Differences compared to no injection p < 0.05 (via paired t test) -122- This paper size applies Chinese national standards ( CNS) A4 specification (210X297mm j I 11 pack-111 I order (please read the precautions on the back before filling out this page) 1221482 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A 7 ____ B7 V. Description of the invention ( 120) Systemic effects of C, muOPG 22-401] -Fc in growing mice. Male BDF1 mice 3-4 weeks old and weighing 9.2-15.7 g were divided into groups of ten. These mice were injected subcutaneously with saline or muOPG [22-401] -Fc 2.5 g / kg twice a day for 14 days (5 mg / kg / day). Before treatment, on days 7 and 14 These mice were radiographed. The mice were sacrificed 24 hours after the last injection. The right thigh was removed, fixed in zinc mafulin, # 5 was removed in formic acid and embedded in the stone core. Sections were cut out from the central region and the femoral axis. They were extended from the metaphyseal end of the growth plate through the primary and secondary bone cancellous to the six adjacent femoral (axis) Histomorphology was used to determine bone density in each area. Each area was 0.5 × 0.5 square millimeters. Changes in radiographs were taken seven days after treatment. Compared with the control group, growth plate associations were seen in OPG-treated mice. Signs of increased bone density in the osteoporotic bone can be seen. This effect is particularly prominent in the distal femur and the proximal tibia (Figure 24A-24B). However, in the vertebral bodies, the iliac crest and the distal tibia also show increased density. By the 14th day, the opaque area has been extended into the thigh and tibia, although the intensity of radiopacity has weakened. In addition, the length of the thigh was not different when the experiment was completed or the experiment was in progress The change in length is also not different, meaning that 0PG does not alter bone growth. Histological changes The distal femoral metaphysis shows increased bone density within 1.1 to 2.65 mm from the growth plate (Figures 25 and 26A- 26B). This is the area where the bone was quickly removed by osteoblast-mediated osteoblast removal in mice. In these fast-growing young rats, it was observed that the bone was treated with OPG in this area. Increase Phenomenon and Bone — ______- 123- Applicable to this paper China National Standard (CNS) A * Specification (21〇 > < 297mm) (Please read the precautions on the back before filling out this page) A7 B7 V. Description of the invention (121) The inhibition of aspiration is consistent. D. The effect of osteopeptide on bone loss caused by ovariectomy in mice will be twelve-week-old female Fisher rats ) Ovariectomy (OVX) or sham surgery was performed and bone density of the distal metaphysis was measured by double X-ray absorptiometry (DEXA). After the three-day recovery period, during the 14-day period, the animals received the following daily injections: ten sham animals received a vehicle injection (phosphate buffered saline); six OVX animals received OPG-Fc 5 mg / Kg subcutaneous injection; six OVX animals received 40 μg / kg estrogen (ESTR) subcutaneous injection. Seven days and 14 days after treatment, the bone density of the animals was measured with DEXA. Two days after the last injection, the animals were sacrificed and the right tibia and joints were excised for histological evaluation. Bone mineral density DEXA measurement showed a tendency for bone density to decrease by OPG-Fc after ovariectomy. The effect is similar to the known anti-absorbent estrogen and pamidronate (Figure 27). Histomorphometric analysis determined that these observations obtained with OPG-F c treatment resulted in significantly higher bone density in OVX mice than in untreated 0 VX mice (Figure 28). These results establish the activity of OPG in bone loss associated with endogenous estrogen withdrawal after ovariectomy. In vivo overview The in vivo effect of recombinant OPG is similar to the changes seen with OPG into foreign gene mice. The reduction in the number of osteoclasts seen in OPG-introduced foreign gene mice can be reproduced by local injection of recombinant OPG in the tops of normal mice and mice treated with IL1-_ or IL1-Lu. OPG introduction of foreign genes in mice will develop petrified petrochemical phenotype, bone marrow cavity will be gradually filled with bone and from the first day after birth will be exhibited from the growth plate unreshaped -124- paper size Applicable to China National Standard (CNS) A4 specification (210X297 mm) --------- install ------ order ----- ^ line (please read the precautions on the back before filling this page) ) 1221482 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (122) Cartilage. For normal three-week-old (growing) mice, OPG treatment also results in the preservation of bone and unremodeled cartilage in the bone formation zone within the cartilage, an effect observed by radiography and determined histologically . Therefore, recombinant OPG will produce phenotypic changes in normal animals similar to those seen in animals introduced with foreign genes, and this change is consistent with the suppression of bone resorption induced by OPG · G. According to the in vitro osteoclast formation assay, a significant part of this inhibition is due to the damaged osteoclast formation. Consistent with this hypothesis, OPG blocks osteoporosis in mice induced by ovariectomy. The bone loss in this model is known to be caused by activated osteoclasts, thus implying the role of OPG in the treatment of primary osteoporosis. Example 1 2 Preparation of N-terminal PEG-OPG complex by pegylated derivative of OPG via reductive alkylation HuOPG met [22-194] P25A was buffer-exchanged to 25-50 mM NaOAc, pH 4.5-4.8 And concentrated to 2-5 mg / ml. This solution was used for OPG reductive alkylation with monofunctional PEG aldehydes at 5-7 ° C. A linear or branched PEG monofunctional aldehyde (available from Shearwater Polymers) with a MW of 1 to 57 kDa (available from Shearwater Polymers) was added to the OPG solution in an amount that could constitute 2-4 mole PEG knee / mol OPG. After the polymer was dissolved in the protein solution, sodium cyanoborohydride was added to the reaction mixture to a final concentration of 15 to 20 mM using a freshly prepared stock solution in cold deionized water of 1-1.6 M. Size exclusion HPLC was performed by elution on a G3000SWXL column (Toso Haas) with 100 mM NaP04, 0.5 M NaCl, 10 ° / 〇 ethanol, pH 6.9 to monitor the progress of the reaction and OPG pegylated Cheng-125- This paper size applies to China National Standard (CNS) Α4 specification (210 × 297 mm) ----------- (Please read the precautions on the back before filling this page)

、1T 1221482 A7 ___B7__ 五、發明説明（123) 度。典型地該反應要進行16-18小時，其後，將反應混合物稀釋6-8倍並將pH降低到3.5-4。將反應混合物以離子交換層析術（HP SP HiLoad 16/10，Pharmacia)用 20 mM NaOAc pH 4以2 5管柱體積，3 0立方米/小時的流速洗提到線型梯度 0.75M NaCl予以分段分離。將含有單一，二或多-聚乙二醇化OPG的洗提份合併並以SEC HPLC和SDS-PAGE鑑定之。經由N-端定序測定出於大部情況中爲主要反應產物的單PEG-OPG拼合物爲98% N_端PEG·改質OPG。此程序通常是用來製備下列N ·端PEG-OPG拼合物（此處的 OPG % HuOPG met [22-194] P25A : 5kD monoPEG，10kD mono 分支 PEG，12 kD monoPEG，20 kD monoPEG，20 kD mono 分支 PEG，25 kD monoPEG，31 kD monoPEG，57 kD monoPEG，12 kD diPEG，25 kD diPEG，31 kD diPEG，57 kD diPEG，25 kD triPEG 0 經由醯基化製備PEG-OPG拼合物經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 將 HuOPG met [22-194] P25A緩衝交換到 50 mM BICINE 緩衝液，pH 8中並濃縮到2 - 3毫克/毫升。因此溶液以單官能 PEG N-羥基丁二醯亞胺基酯在室溫下進行OPG醯化。將線型或分枝，MW=1至57 kDa的PEG N_羥基丁二醯亞胺基酯 (可得自Shearwater Polymers)以固體形式可達到4-8莫耳PEG N-羥基丁二醯亞胺基酯/莫耳OPG之量加到OPG溶液内。用尺寸排斥 HPLC 在 G3000SWXL 管柱（Toso Haas)上用 100 mM MaP04，0.5 M NaCl，10%乙醇，pH 6.9洗提以監測反應的進展和OPG PEG化之程度。典型地，該反應要進行1小時， -126- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X297公釐） 1221482 經濟部中央榡準局員工消費合作衽印製 A7 B7_ _ 五、發明説明（124) 其後將反應混合物稀釋6-8倍並將pH降到3.5-4。將反應混合物以離子交換層析術（HP SP HiLoad 16/10, Pharmacia)用20 mM NaOAc pH4，於2 5管柱體積以3 0立方米/小時流速到 0·75 M NaCl之線型梯度洗提分段分離。將單-，雙-或多-PEG化OPG的洗提液份合併並用SEC HPLC和SDS-PAGE予以鑑定。此程序通用地用來製備下列PEG-OPG拼合物：5 kD多 PEG，20 kD 多 PEG，40 kD 多分枝PEG，50 kD 多 PEG。三體物PEG-OPG之製備將HuOPG met[22-194] P25A置於近乎中性pH的磷酸鹽緩衝液中以1 ·3毫克/毫升濃度準備硫醇化之用。於保持pH在7.0 之下，將S-乙醯基氫硫基丁二酸酐（AMSA)以3-7倍莫耳超量加入並在4 °C下將反應攪拌2小時。以離子交換層析術從未改質和多硫醇化OPG中分離出單硫醇化-OPG並用羥胺處理以脱除經保護硫醇的保護基。於去保護之後，以凝膠過濾去除羥胺並對所得單硫醇化· OPG施以各種硫醇特定*** 聯化學。爲了產生雙硫鍵結二體物，與以>1毫克/毫升的硫醇化OPG經由透析到微鹼性磷酸鹽緩衝液中進行空氣氧化。經由用雙順丁烯二醯亞胺交聯劑，N，N-雙（3 -順丁烯二酿亞胺基丙醯基）_2_羥基1，3丙烷與該硫醇化0PG(>1毫克/毫升）於0.6乂交聯劑：〇?〇莫耳比例下在？116.5磷酸鹽緩衝液中反應而製備共價硫醚OPG二體物。類似地，經由用次化學計算量的雙-順丁烯二醯亞安PEG交聯劑與> 1毫克/毫升硫醇化 OPG在磷酸鹽緩衝液pH 6.5内反應而製成PEG亞铃。任何種 L _-127- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ---------------IT-----N 線 (請先閲讀背面之注意事項再填寫本頁) 1221482 A7 B7 五、發明説明（127) 序列表 (1) 一般資訊： (i) 申請者·· Amgen Inc. (ii) 發明名稱：促骨堆積肽 (iii) 序列數目：168 (iv) 通信地址： (A) 收件人：Amgen Inc. (B) 街名：1840 Dehavilland Drive (C) 市名：Thousand Oaks (D) 州名：加州 (E) 國名：美國 (F) 郵遞區號：91320 (v) 計算機可讀形式： (A) 媒體類別：軟磁片 (B) 計算機：IBM PC相容, 1T 1221482 A7 ___B7__ 5. Description of the invention (123) degrees. The reaction is typically carried out for 16-18 hours, after which the reaction mixture is diluted 6-8 times and the pH is lowered to 3.5-4. The reaction mixture was washed by ion exchange chromatography (HP SP HiLoad 16/10, Pharmacia) with 20 mM NaOAc pH 4 at a column volume of 25 at a flow rate of 30 m3 / h and separated into a linear gradient of 0.75 M NaCl. Segment separation. The fractions containing single, di- or poly-PEGylated OPG were combined and identified by SEC HPLC and SDS-PAGE. The N-terminal sequencing determined that the mono-PEG-OPG complex, which is the main reaction product in most cases, was 98% N-terminal PEG-modified OPG. This procedure is usually used to prepare the following N-terminal PEG-OPG complexes (here OPG% HuOPG met [22-194] P25A: 5kD monoPEG, 10kD mono branched PEG, 12 kD monoPEG, 20 kD monoPEG, 20 kD mono Branched PEG, 25 kD monoPEG, 31 kD monoPEG, 57 kD monoPEG, 12 kD diPEG, 25 kD diPEG, 31 kD diPEG, 57 kD diPEG, 25 kD triPEG 0 Preparation of PEG-OPG complex via amidation Printed by the employee consumer cooperative (please read the notes on the back before filling out this page). Exchange HuOPG met [22-194] P25A buffer into 50 mM BICINE buffer, pH 8 and concentrate to 2-3 mg / ml. Therefore The solution was OPG-fluorinated with monofunctional PEG N-hydroxysuccinimide ester at room temperature. Linear or branched PEG N-hydroxysuccinimide ester (can be (From Shearwater Polymers) was added to the OPG solution in an amount of 4-8 moles of PEG N-hydroxysuccinimide / mole OPG in solid form. Size-exclusion HPLC on a G3000SWXL column (Toso Haas) Elution with 100 mM MaP04, 0.5 M NaCl, 10% ethanol, pH 6.9 to monitor the progress of the reaction The degree of OPG PEGylation. Typically, the reaction takes one hour. -126- This paper size applies the Chinese National Standard (CNS) A4 specification (21 × 297 mm). Preparation of A7 B7_ _ V. Description of the invention (124) The reaction mixture was then diluted 6-8 times and the pH was lowered to 3.5-4. The reaction mixture was used for ion exchange chromatography (HP SP HiLoad 16/10, Pharmacia) 20 mM NaOAc pH 4 was separated in a 25-column column with a linear gradient elution of 30 cubic meters per hour at a flow rate of 0 · 75 M NaCl. Single-, double-, or multi-PEGylated OPG eluent Fractions were combined and identified by SEC HPLC and SDS-PAGE. This procedure is commonly used to prepare the following PEG-OPG complexes: 5 kD polyPEG, 20 kD polyPEG, 40 kD multibranched PEG, 50 kD polyPEG. Trisomy Preparation of PEG-OPG HuOPG met [22-194] P25A was prepared in a near neutral pH phosphate buffer solution for thiolation at a concentration of 1.3 mg / ml. While maintaining the pH below 7.0, S-acetamidohydrothiosuccinic anhydride (AMSA) was added at a molar amount of 3-7 times and the reaction was stirred at 4 ° C for 2 hours. Monothiolated-OPG was separated from the unmodified and polythiolated OPG by ion exchange chromatography and treated with hydroxylamine to remove the protecting thiol protecting group. After deprotection, hydroxylamine was removed by gel filtration, and various monothiol-specific cross-linking chemistry was applied to the resulting monothiolized OPG. To produce a disulfide-bonded dimer, air oxidation with thiolated OPG at > 1 mg / ml was performed by dialyzing into a slightly alkaline phosphate buffer. By using a bis-cis-butene-diimine cross-linking agent, N, N-bis (3-cis-butene-diimidopropionyl) _2_hydroxy1,3propane is reacted with the thiolated 0PG (> 1 Mg / ml) at 0.6 乂 Crosslinker: 〇〇〇mole ratio? Reaction in 116.5 phosphate buffer to prepare covalent thioether OPG dimer. Similarly, a PEG subbell was made by reacting a sub-stoichiometric amount of bis-cis-butenedifluorene PEG crosslinker with > 1 mg / ml thiolated OPG in a phosphate buffer pH 6.5. Any kind of L _-127- This paper size is applicable to China National Standard (CNS) A4 specification (210X297mm) --------------- IT ----- N line (please first Read the notes on the back and fill in this page) 1221482 A7 B7 V. Description of the invention (127) Sequence listing (1) General information: (i) Applicant · Amgen Inc. (ii) Name of invention: Osteoportic peptide (iii ) Number of Sequences: 168 (iv) Mailing Address: (A) Recipient: Amgen Inc. (B) Street Name: 1840 Dehavilland Drive (C) City Name: Thousand Oaks (D) State Name: California (E) Country Name : United States (F) ZIP Code: 91320 (v) Computer-readable form: (A) Media category: Flexible disk (B) Computer: IBM PC compatible

(C) 操作系統：PC-DOS/MS-DOS (D) 軟體：Patentln Release #1.0，Version #1.30 (vi) 現行申請數據： (A) 申請編號：經濟部中央標準局員工消費合作社印製 (B) 提申曰期： (C) 分類： (viii)代理律師/代理人： (A) 名稱：Winter，Robert B. (B) 參考文件檔號：A-3 78_CIP2 -130- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（128) (2) SEQ ID NO : 1的資訊： (i) 序列特性： (A) 長度：36鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(C) Operating system: PC-DOS / MS-DOS (D) Software: Patentln Release # 1.0, Version # 1.30 (vi) Current application data: (A) Application number: Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ( B) Date of application: (C) Classification: (viii) Attorney / Attorney: (A) Name: Winter, Robert B. (B) Reference file number: A-3 78_CIP2 -130- This paper standard applies China National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 V. Description of the invention (128) (2) Information of SEQ ID NO: 1: (i) Sequence characteristics: (A) Length: 36 base pairs (B ) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 1 : AAAGGAAGGA AAAAAGCGGC CGCTACANNN NNNNNT 36 (2) SEQ ID NO : 2的資訊： (i) 序列特性： (A) 長度：16鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 1: AAAGGAAGGA AAAAAGCGGC CGCTACANNN NNNNNT 36 (2) Information of SEQ ID NO: 2: (i) Sequence characteristics: (A) Length: 16 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 2 : TCGACCCACG CGTCCG 16 (2) SEQ ID NO : 3的資訊： (i) 序列特性： (A) 長度：12鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 2: TCGACCCACG CGTCCG 16 (2) Information of SEQ ID NO: 3: (i) Sequence characteristics: (A) Length: 12 base pairs (B) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA 經濟部中央標準局員工消費合作社印製 (xi)序列説明：SEQ ID NO : 3 : GGTGCGCAG GC 12 (2) SEQ ID NO : 4的資訊·· (i)序列特性： (A) 長度：18鹼對 (B) 類別：核酸 (C) 股型··單股 -131 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（129) (D)拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 4 : TGTAAAACGA CGGCCAGT 18 (2) SEQ ID NO : 5的貴訊： (i) 序列特性： (A) 長度：18鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (xi) Sequence description: SEQ ID NO: 3: GGTGCGCAG GC 12 (2) Information of SEQ ID NO: 4 (i) Sequence characteristics: (A) Length: 18 alkali pairs (B) Category: Nucleic acid (C) Strand type ·· Single-strand -131-This paper size applies to China National Standard (CNS) A4 (210X 297 mm) 1221482 Central Bureau of Standards, Ministry of Economic Affairs Printed by employee consumer cooperative A7 B7 V. Description of the invention (129) (D) Topology: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 4: TGTAAAACGA CGGCCAGT 18 (2) SEQ ID NO: 5 of your news: (i) sequence characteristics: (A) length: 18 base pairs (B) category: nucleic acid (C) strand type: single strand (D) topology: linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 5 : CAGGAAACAG CTATGACC 18 (2) SEQ ID NO : 6的資訊·· (i) 序列特性： (A) 長度：20鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 5: CAGGAAACAG CTATGACC 18 (2) Information of SEQ ID NO: 6 (i) Sequence characteristics: (A) Length: 20 base pairs (B ) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 6 : CAATTAACCC TCACTAAAGG 20 (2) SEQ ID NO : 7的資訊： (i) 序列特性： (A) 長度：23鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 6: CAATTAACCC TCACTAAAGG 20 (2) Information of SEQ ID NO: 7: (i) Sequence characteristics: (A) Length: 23 base pairs (B) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 7 : GCATTATGAC CCAGAAACCG GAC 23 __-132- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 裝. 1221482 經濟部中央標準局員工消費合作社印製 A7 B7五、發明説明（130) (2) SEQ ID NO : 8的資訊： (i) 序列特性： (A) 長度：2 3驗對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型 (ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 8 : AGGTAGCGCC CTTCCTCACA TTC 23 (2) SEQ ID NO : 9的資訊： (i) 序列特性： (A) 長度：3 0驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 9 : GACTAGTCCC ACAATGAACA AGTGGCTGTG 30 (2) SEQ ID NO : 10的資訊： (i) 序列特性： (A) 長度：45鹼對 (B) 類別：核酸(C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 10 : ATAAGAATGC GGCCGCTAAA CTATGAAACA GCCCAGTGAC CATTC 45 (2) SEQ ID NO : 11的資訊： (i)序列特性： (A) 長度：2 1鹼對 (B) 類別：核酸 (C) 股型：單股 (請先閱讀背面之注意事項再填寫本頁) -133- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（131) (D)拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 11 : GCCTCTAGAA AGAGCTGGGA C 21 (2) SEQ ID NO : 12的資訊： (i) 序列特性： (A) 長度：21鹼對 (B) 類別：核酸 (C) 股型。·單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 7: GCATTATGAC CCAGAAACCG GAC 23 __- 132- This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the back Note: Please fill in this page again) Pack. 1221482 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Description (130) (2) Information of SEQ ID NO: 8: (i) Sequence characteristics: (A) Length : 2 3 Test pair (B) Category: Nucleic acid (C) Strand type ·· Single strand (D) Topology: Linear type (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 8: AGGTAGCGCC CTTCCTCACA TTC 23 (2) Information of SEQ ID NO: 9: (i) Sequence characteristics: (A) Length: 30 Check pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear type (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 9: GACTAGTCCC ACAATGAACA AGTGGCTGTG 30 (2) Information of SEQ ID NO: 10: (i) Sequence characteristics: (A) Length: 45 base pairs (B) Category: Nucleic acid (C) strand type: single strand (D) topology: linear type (ii) molecular class: cDNA (xi) sequence description: SEQ ID NO: 10: ATAAGAATGC GGCCGCTAAA CTAT GAAACA GCCCAGTGAC CATTC 45 (2) Information of SEQ ID NO: 11: (i) Sequence characteristics: (A) Length: 2 1 Alkali pair (B) Category: Nucleic acid (C) Strand type: Single strand (Please read the back Please fill in this page again for the matters needing attention) -133- This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297mm) 1221482 A7 B7 V. Description of the invention (131) (D) Topology: Linear (ii) Molecular category: cDNA (xi) sequence description: SEQ ID NO: 11: GCCTCTAGAA AGAGCTGGGA C 21 (2) Information of SEQ ID NO: 12: (i) Sequence characteristics: (A) Length: 21 base pairs (B) Category: Nucleic acid (C ) Stock type. · Single strand (D) Topology: Linear

(ii) 分子類別·· cDNA (xi)序列説明：SEQ ID NO : 12 : CGCCGTGTTC CATTTATGAG C 21 (2) SEQ ID NO : 13的資訊： (i) 序列特性： (A) 長度：24鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type · cDNA (xi) Sequence description: SEQ ID NO: 12: CGCCGTGTTC CATTTATGAG C 21 (2) Information of SEQ ID NO: 13: (i) Sequence characteristics: (A) Length: 24 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明·· SEQ ID NO : 13 ·· ATCAAAGGCA GGGCATACTT CCTG 24 (2) SEQ ID NO : 14的資訊： (i) 序列特性： (A) 長度：24鹼對經濟部中央標準局員工消費合作社印製 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description · SEQ ID NO: 13 · ATCAAAGGCA GGGCATACTT CCTG 24 (2) Information of SEQ ID NO: 14: (i) Sequence characteristics: (A) Length: 24 base pairs Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (B) Category: Nucleic Acid (C) Stock Type: Single Stock (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 14 : GTTGCACTCC TGTTTCACGG TCTG 24 -134- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（132) (2) SEQ ID NO : 15的資訊 0 (i) 序列特性： (A) 長度：24鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 14: GTTGCACTCC TGTTTCACGG TCTG 24 -134- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 V. Description of the invention (132) (2) Information of SEQ ID NO: 15 0 (i) Sequence characteristics: (A) Length: 24 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 15 : CAAGACACCT TGAAGGGCCT GATG 24 (2) SEQ ID NO : 16的資訊： (i) 序列特性： (A) 長度：24鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 15: CAAGACACCT TGAAGGGCCT GATG 24 (2) Information of SEQ ID NO: 16: (i) Sequence characteristics: (A) Length: 24 base pairs (B ) Category: Nucleic Acid (C) Strand Type ·· Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 16 : TAACTTTTAC AGAAGAGCAT CAGC 24 (2) SEQ ID NO : 17的資訊： (i) 序列特性： (A) 長度：33鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 16: TAACTTTTAC AGAAGAGCAT CAGC 24 (2) Information of SEQ ID NO: 17: (i) Sequence characteristics: (A) Length: 33 base pairs (B ) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別·· cDNA 經濟部中央標準局員工消費合作社印製 (xi)序列説明：SEQ ID NO : 17 : AGCGCGGCCG CATGAACAAG TGGCTGTGCT GCG 33 (2) SEQ ID NO ·· 18的資訊·· (i)序列特性： (A) 長度：3 1鹼對 (B) 類別：核酸 (C) 股型：單股 -135- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 2 8 4 11 2 12 A7 B7 五、發明説明（133) (D)拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 18 : AGCTCTAGAG AAACAGCCCA GTGACCATTC C 31 (2) SEQ ID NO : 19的資訊： (i) 序列特性： (A) 長度：24鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學··線型(ii) Molecular type · cDNA Printed by the Consumer Cooperative of the Central Standard Bureau of the Ministry of Economic Affairs (xi) Sequence description: SEQ ID NO: 17: AGCGCGGCCG CATGAACAAG TGGCTGTGCT GCG 33 (2) Information of SEQ ID NO · · 18 ·· (i ) Sequence characteristics: (A) Length: 3 1 Alkali pair (B) Category: Nucleic acid (C) Stock type: Single-135- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 2 8 4 11 2 12 A7 B7 V. Description of the invention (133) (D) Topology: linear (ii) molecular type: cDNA (xi) sequence description: SEQ ID NO: 18: AGCTCTAGAG AAACAGCCCA GTGACCATTC C 31 (2) SEQ ID NO: Information of 19: (i) Sequence characteristics: (A) Length: 24 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology ·· Linearity

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 19 : GTGAAGCTGT GCAAGAACCT GATG 24 (2) SEQ ID NO : 20的資訊： (i) 序列特性： (A) 長度：24鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 19: GTGAAGCTGT GCAAGAACCT GATG 24 (2) Information of SEQ ID NO: 20: (i) Sequence characteristics: (A) Length: 24 base pairs (B ) Category: Nucleic Acid (C) Strand Type ·· Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 20 : ATCAAAGGCA GGGCATACTT CCTG 24 (2) SEQ ID NO : 21的資訊： (i) 序列特性： (A) 長度：24鹼對經濟部中央標準局員工消費合作社印製 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 20: ATCAAAGGCA GGGCATACTT CCTG 24 (2) Information of SEQ ID NO: 21: (i) Sequence characteristics: (A) Length: 24 Printed by the Central Standards Bureau Consumer Cooperative (B) Category: Nucleic Acid (C) Stock Type: Single Stock (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 21 : CAGATCCTGA AGCTGCTCAG TTTG 24 -136· 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（134) (2) SEQ ID NO : 22的資訊： (i) 序列特性： (A) 長度：33鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學··線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 21: CAGATCCTGA AGCTGCTCAG TTTG 24 -136 · This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 V. Description of the invention (134) (2) Information of SEQ ID NO: 22: (i) Sequence characteristics: (A) Length: 33 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology ·· Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 22 : AGCGCGGCCG CGGGGACCAC AATGAACAAG TG 33 (2) SEQ ID NO : 23的資訊·· (i) 序列特性： (A) 長度：33鹼對 (B) 類別··核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 22: AGCGCGGCCG CGGGGACCAC AATGAACAAG TG 33 (2) Information of SEQ ID NO: 23 ... (i) Sequence characteristics: (A) Length: 33 base pairs (B) Type ·· Nucleic Acid (C) Strand: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明·· SEQ ID NO : 23 : AGCTCTAGAA TTGTGAGGAA ACAGCTCAAT GGC 33 (2) SEQ ID NO : 24的資訊： (i) 序列特性： (A) 長度：39鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description. SEQ ID NO: 23: AGCTCTAGAA TTGTGAGGAA ACAGCTCAAT GGC 33 (2) Information of SEQ ID NO: 24: (i) Sequence characteristics: (A) Length: 39 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA 經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) (xi)序列説明：SEQ ID NO : 24 : ATAGCGGCCG CTGAGCCCAA ATCTTGTGAC AAAACTCAC 39 (2) SEQ ID NO : 25的資訊： (i)序列特性： (A) 長度：45鹼對 (B) 類別：核酸 (C) 股型：單股 -137- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 經濟部中央標準局員工消費合作社印製 B7___ 五、發明説明（135) (D)拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 25 : TCTAGAGTCG ACTTATCATT TACCCGGAGA GAGGGAGAGG CTCTT 45 (2) SEQ ID NO : 26的資訊： (i) 序列特性： (A) 長度：3 8鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) (xi) Sequence description: SEQ ID NO: 24: ATAGCGGCCG CTGAGCCCAA ATCTTGTGAC AAAACTCAC 39 (2) Information of SEQ ID NO: 25: (i) Sequence characteristics: (A) Length: 45 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand -137- This paper size applies Chinese National Standard (CNS) A4 Specifications (210X297 mm) 1221482 A7 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs B7___ V. Description of the invention (135) (D) Topology: Linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 25: TCTAGAGTCG ACTTATCATT TACCCGGAGA GAGGGAGAGG CTCTT 45 (2) Information of SEQ ID NO: 26: (i) Sequence characteristics: (A) Length: 3 8 base pairs (B) Category: Nucleic acid (C) Type of share: Single ) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 26 : CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) SEQ ID NO : 27的資訊： (i) 序列特性： (A) 長度：43鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 26: CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) Information of SEQ ID NO: 27: (i) Sequence characteristics: (A) Length: 43 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明·· SEQ ID NO : 27 ·· CCTCTGCGGC CGCTAAGCAG CTTATTTTCA CGGATTGAAC CTG 43 (2) SEQ ID NO : 28的資訊： (i) 序列特性： (A) 長度：38鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description · SEQ ID NO: 27 · CCTCTGCGGC CGCTAAGCAG CTTATTTTCA CGGATTGAAC CTG 43 (2) Information of SEQ ID NO: 28: (i) Sequence characteristics: (A) Length: 38 Base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 28 : CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 __-138- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） ----------裝-- (請先閱讀背面之注意事項再填寫本頁) 訂 1221482 A7 B7 五、發明説明（13β) (2) SEQ ID NO : 29的資訊： (i) 序列特性： (A) 長度·· 2 4驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 28: CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 __- 138- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) ---- ------ Install-(Please read the notes on the back before filling this page) Order 1221482 A7 B7 V. Description of the invention (13β) (2) Information of SEQ ID NO: 29: (i) Sequence characteristics: (A) Length ·· 2 4 Test pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明·· SEQ ID NO ·· 29 ·· TCCGTAAGAA ACAGCCCAGT GACC 24 (2) SEQ ID NO : 30的資訊·· (i) 序列特性： (A) 長度：3 1鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description. SEQ ID NO. 29. TCCGTAAGAA ACAGCCCAGT GACC 24 2. Information of SEQ ID NO: 30. (i) Sequence characteristics: (A) Length: 3 1 Alkali pair (B) Category: Nucleic acid (C) Strand type · · Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 30 : CCTCTGCGGC CGCTGTTGCA TTTCCTTTCT G 31 (2) SEQ ID NO : 31 的資訊： (i) 序列特性： (A) 長度：19胺基酸 (B) 類別：胺基酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：蛋白質經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) (xi)序列説明：SEQ ID NO : 31 :(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 30: CCTCTGCGGC CGCTGTTGCA TTTCCTTTCT G 31 (2) Information of SEQ ID NO: 31: (i) Sequence characteristics: (A) Length: 19 amino acid (B) Type: Amino acid (C) Stock type: Single stock (D) Topology: Linear type (ii) Molecular type: Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Protein Economy (Please read the notes on the back before filling out (This page) (xi) Sequence description: SEQ ID NO: 31:

Glu Thr Leu Pro Pro Lys Tyr Leu His Tyr Asp Pro Glu Thr Gly His 15 10 15Glu Thr Leu Pro Pro Lys Tyr Leu His Tyr Asp Pro Glu Thr Gly His 15 10 15

Gin Leu Leu (2) SEQ ID NO : 32的資訊： (i)序列特性： _-139- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（137) (A) 長度：2 1鹼對 (B) 類別：核酸 (C) 股型：單股Gin Leu Leu (2) Information of SEQ ID NO: 32: (i) Sequence characteristics: _-139- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 V. Description of the invention (137 ) (A) Length: 2 1 Base pairs (B) Category: Nucleic acid (C) Strand type: Single strand

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 32 : TCCCTTGCCC TGACCACTCT T 21 (2) SEQ ID NO : 33的資訊： (i) 序列特性： (A) 長度：34鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 32: TCCCTTGCCC TGACCACTCT T 21 (2) Information of SEQ ID NO: 33: (i) Sequence characteristics: (A) Length: 34 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 33 : CCTCTGCGGC CGCACACACG TTGTCATGTG TTGC 34 (2) SEQ ID NO : 34的資訊： (i) 序列特性： (A) 長度：21鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 33: CCTCTGCGGC CGCACACACG TTGTCATGTG TTGC 34 (2) Information of SEQ ID NO: 34: (i) Sequence characteristics: (A) Length: 21 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 34 : TCCCTTGCCC TGACCACTCT T 21 經濟部中央標準局員工消費合作社印製 (2) SEQ ID NO : 35的資訊： (i) 序列特性： (A) 長度：34鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 34: TCCCTTGCCC TGACCACTCT T 21 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (2) Information of SEQ ID NO: 35: (i) Sequence characteristics: (A) Length: 34 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA -140- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 ___ _B7 五、發明説明（138) (xi)序列説明：SEQ ID NO ·· 35 ·· CCTCTGCGGC CGCCTTTTGC GTGGCTTCTC TGTT 34 (2) SEQ ID NO : 36的資訊： (i) 序列特性： (A) 長度：3 7鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA -140- This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) 1221482 Printed by A7 of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs _ _B7 V. Description of the invention (138) (xi ) Sequence description: SEQ ID NO ·· 35 ·· CCTCTGCGGC CGCCTTTTGC GTGGCTTCTC TGTT 34 (2) Information of SEQ ID NO: 36: (i) Sequence characteristics: (A) Length: 37 Base pairs (B) Category: Nucleic acid ( C) Stock Type: Single Stock (D) Topology: Linear

(ii) 分子類別·· cDNA (xi)序列説明：SEQ ID NO : 36 : CCTCTGAGCT CAAGCTTGGT TTCCGGGGAC CACAATG 37 (2) SEQ ID NO : 37的資訊： (i) 序列特性： (A) 長度：38鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class · cDNA (xi) Sequence description: SEQ ID NO: 36: CCTCTGAGCT CAAGCTTGGT TTCCGGGGAC CACAATG 37 (2) Information of SEQ ID NO: 37: (i) Sequence characteristics: (A) Length: 38 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 37 : CCTCTGCGGC CGCTAAGCAG CTTATTTTTA CTGAATGG 38 (2) SEQ ID NO : 38的資訊： (i) 序列特性： (A) 長度：37鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 37: CCTCTGCGGC CGCTAAGCAG CTTATTTTTA CTGAATGG 38 (2) Information of SEQ ID NO: 38: (i) Sequence characteristics: (A) Length: 37 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 38 : CCTCTGAGCT CAAGCTTGGT TTCCGGGGAC CACAATG 37 (2) SEQ ID NO : 39的資訊： (i)序列特性： -141 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ---------f ^------、p------^綉 (請先閲讀背面之注意事項再填寫本頁) 1221482 A7 B7 五、發明説明（139) (A) 長度：33鹼對 (B) 類別：核酸 (C) 股型：單股(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 38: CCTCTGAGCT CAAGCTTGGT TTCCGGGGAC CACAATG 37 (2) Information of SEQ ID NO: 39: (i) Sequence characteristics: -141-This paper is for Chinese countries Standard (CNS) A4 specification (210X297 mm) --------- f ^ ------, p ------ ^ embroidery (Please read the precautions on the back before filling this page ) 1221482 A7 B7 V. Description of the invention (139) (A) Length: 33 base pairs (B) Category: Nucleic acid (C) Stock type: Single strand

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 39 : CCTCTGCGGC CGCCAGGGTA ACATCTATTC CAC 33 (2) SEQ ID NO : 40的資訊： (i) 序列特性： (A) 長度：35鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 39: CCTCTGCGGC CGCCAGGGTA ACATCTATTC CAC 33 (2) Information of SEQ ID NO: 40: (i) Sequence characteristics: (A ) Length: 35 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 40 : CCGAAGCTTC CACCATGAAC AAGTGGCTGT GCTGC 35 (2) SEQ ID NO : 41的資訊： (i) 序列特性： (A) 長度：40鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 40: CCGAAGCTTC CACCATGAAC AAGTGGCTGT GCTGC 35 (2) Information of SEQ ID NO: 41: (i) Sequence characteristics: (A) Length: 40 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 41 : CCTCTGTCGA CTATTATAAG CAGCTTATTT TCACGGATTG 40 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) (2) SEQ ID NO : 42的資訊： (i) 序列特性： (A) 長度：21鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 41: CCTCTGTCGA CTATTATAAG CAGCTTATTT TCACGGATTG 40 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) (2) Information about SEQ ID NO: 42: (i) Sequence characteristics: (A) Length: 21 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA -142- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（140) (xi)序列説明：SEQ ID NO : 42 : TCCCTTGCCC TGACCACTCT T 21 (2) SEQ ID NO : 43的資訊： (i) 序列特性： (八）長度：35鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA -142- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 V. Description of the invention (140) (xi) Sequence description: SEQ ID NO: 42: TCCCTTGCCC TGACCACTCT T 21 (2) Information of SEQ ID NO: 43: (i) Sequence characteristics: (eight) Length: 35 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear type

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 43 : CCTCTGTCGA CTTAACACAC GTTGTCATGT GTTGC 35 (2) SEQ ID NO : 44的資訊： (i) 序列特性： (A) 長度：21鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 43: CCTCTGTCGA CTTAACACAC GTTGTCATGT GTTGC 35 (2) Information of SEQ ID NO: 44: (i) Sequence characteristics: (A) Length: 21 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 44 : TCCCTTGCCC TGACCACTCT T 21 (2) SEQ ID NO : 45的資訊： (i) 序列特性： (A) 長度：35鹼對 (B) 類別：核酸 (C) 股型：單股經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 44: TCCCTTGCCC TGACCACTCT T 21 (2) Information of SEQ ID NO: 45: (i) Sequence characteristics: (A) Length: 35 base pairs (B ) Category: Nucleic acid (C) Stock type: Single-share printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 45 : CCTCTGTCGA CTTACTTTTG CGTGGCTTCT CTGTT 35 (2) SEQ ID NO : 46的資訊： (i)序列特性： -143- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（Μ1) (A) 長度：1 53 7鹼對 (B) 類別：核酸 (C) 股型：單股(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 45: CCTCTGTCGA CTTACTTTTG CGTGGCTTCT CTGTT 35 (2) Information of SEQ ID NO: 46: (i) Sequence characteristics: -143- This paper is applicable to China Standard (CNS) A4 specification (210X297 mm) 1221482 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (M1) (A) Length: 1 53 7 Base pair (B) Category: Nucleic acid (C) Share type: single

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 46 : — .1 I I I I I I I I I I I I I 訂 I I I 線 (請先閲讀背面之注意事項再填寫本頁) -144- GTGAAGAGCG TGAAGAGCGG TTCCTCCTTT CAGCAAAAAA CCCCTCAAGA CCCGTTTAGA 60 GGCCCCAAGG GGTTATGCTA GTTATTGCTC AGCGGTGGCA GCAGCCAACT CAGCTTCCTT 120 TCGGGCTTTC TTCTTCTTCT TCTTCTTTCC GCGGATCCTC GAGTAAGCTT CCATGGTACC 180 CTGCAGGTCG ACACTAGTGA GCTGGAATTC CAACGCGTTA ACCATATGTT ATTCCTCCTT 240 TAATTAGTTA AAACAAATCT AGAATCAAAT CGATTAATCG ACTATAACAA ACCATTTTCT 300 TGCGTAAACC TGTACGATCC TACAGGTACT TATGTTAAAC AATTGTATTT CAAGCGATAT 360 AATAGTGTGA CAAAAATCCA ATTTATTAGA ATCAAATGTC AATCTATTAC GGTTTTAATG 420 ATATATAACA CGCAAAACTT GCGACAAACA ATAGGTAAGG ATAAAGAGAT GGGTATGAAA 480 GACATAAATG CCGACGACAC TTACAGAATA ATTAATAAAA TTAAAGCCTG TAGAAGCAAT 540 AATGATATTA ATCAATGCTT ATCTGATATG ACTAAAATGG TACATTGTGA ATATTATTTA 600 CTCGCGATCA TTTATCCTCA TTCTATGGTT AAATCTGATA TTTCAATTCT GGATAATTAC 660 CCTAAAAAAT GGAGGCAATA TTATGATGAC GCTAATTTAA TAAAATATGA TCCTATAGTA 720 GATTATTCTA ACTCCAATCA TTCACCGATT AATTGGAATA TATTTGAAAA CAATGCTGTA 780 AATAAAAAAT CTCCAAATGT AATTAAAGAA GCGAAATCAT CAGGTCTTAT CACTGGGTTT 840 AGTTTCCCTA TTCATACTGC TAATAATGGC TTCGGAATGC TTAGTTTTGC ACATTCAGAG 900 AAAGACAACT ATATAGATAG TTTATTTTTA CATGCGTGTA TGAACATACC ATTAATTGTT 960 CCTTCTCTAG TTGATAATTA TCGAAAAATA AATATAGCAA ATAATAAATC AAACAACGAT 1020 TTAACCAAAA GAGAAAAAGA ATGTTTAGCG TGGGCATGCG AAGGAAAAAG CTCTTGGGAT 1080 ATTTCAAAAA TATTAGGCTG TAGTAAGCGC ACGGTCACTT TCCATTTAAC CAATGCGCAA 1140 ATGAAACTCA ATACAACAAA CCGCTGCCAA AGTATTTCTA AAGCAATTTT AACAGGAGCA 1200 ATTGATTGCC CATACTTTAA AAGTTAAGTA CGACGTCCAT ATTTGAATGT ATTTAGAAAA 1260 本紙張又度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局員工消費合作社印聚 A7 B7 五、發明説明（142) ATAAACAAAA GAGTTTGTAG AAACGCAAAA AGGCCATCCG TCAGGATGGC CTTCTGCTTA 1320 ATTTGATGCC TGGCAGTTTA TGGCGGGCGT CCTGCCCGCC ACCCTCCGGG CCGTTGCTTC 1380 GCAACGTTCA AATCCGCTCC CGGCGGATTT GTCCTACTCA GGAGAGCGTT CACCGACAAA 1440 CAACAGATAA AACGAAAGGC CCAGTCTTTC GACTGAGCCT TTCGTTTTAT TTGATGCCTG 1500 GCAGTTCCCT ACTCTCGCAT GGGGAGACCA TGCATAC 1537 (2) SEQ ID NO ·· 47的資訊·· (i) 序列特性·· (八）長度：48鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 46: — .1 IIIIIIIIIIIII Order III line (please read the precautions on the back before filling this page) -144- GTGAAGAGCG TGAAGAGCGG TTCCTCCTTT CAGCAAAAAA CCCCTCAAGA CCCGTTTAGA 60 GGCCCCAAGG GGTTATGCTA GTTATTGCTC AGCGGTGGCA GCAGCCAACT CAGCTTCCTT 120 TCGGGCTTTC TTCTTCTTCT TCTTCTTTCC GCGGATCCTC GAGTAAGCTT CCATGGTACC 180 CTGCAGGTCG ACACTAGTGA GCTGGAATTC CAACGCGTTA ACCATATGTT ATTCCTCCTT 240 TAATTAGTTA AAACAAATCT AGAATCAAAT CGATTAATCG ACTATAACAA ACCATTTTCT 300 TGCGTAAACC TGTACGATCC TACAGGTACT TATGTTAAAC AATTGTATTT CAAGCGATAT 360 AATAGTGTGA CAAAAATCCA ATTTATTAGA ATCAAATGTC AATCTATTAC GGTTTTAATG 420 ATATATAACA CGCAAAACTT GCGACAAACA ATAGGTAAGG ATAAAGAGAT GGGTATGAAA 480 GACATAAATG CCGACGACAC TTACAGAATA ATTAATAAAA TTAAAGCCTG TAGAAGCAAT 540 AATGATATTA ATCAATGCTT ATCTGATATG ACTAAAATGG TACATTGTGA GATTACATCAATTCTATTCT 600 CTCGCGATCA TTTAGGCTCTCACATT GCTAATTTAA TAAAATATGA TCCTATAGTA 720 GATTATTCTA ACTCCAATCA TTCACCGATT AATTGGAATA TATTTGAAAA CAATGCTGTA 780 AATAAAAAAT CTCCAAATGT AATTAAAGAA GCGAAATCAT CAGGTCTTAT CACTGGGTTT 840 AGTTTCCCTA TTCATACTGC TAATAATGGC TTCGGAATGC TTAGTTTTGC ACATTCAGAG 900 AAAGACAACT ATATAGATAG TTTATTTTTA CATGCGTGTA TGAACATACC ATTAATTGTT 960 CCTTCTCTAG TTGATAATTA TCGAAAAATA AATATAGCAA ATAATAAATC AAACAACGAT 1020 TTAACCAAAA GAGAAAAAGA ATGTTTAGCG TGGGCATGCG AAGGAAAAAG CTCTTGGGAT 1080 ATTTCAAAAA TATTAGGCTG TAGTAAGCGC ACGGTCACTT TCCATTTAAC CAATGCGCAA 1140 ATGAAACTCA ATACAACAAA CCGCTGCCAA AGTATTTCTA AAGCAATTTT AACAGGAGCA 1200 ATTGATTGCC CATACTTTAA AAGTTAAGTA CGACGTCCAT ATTTGAATGT ATTTAGAAAA 1260 This paper is also compatible with the China National Standard (CNS) A4 Cooperative Bureau of China 4 specifications (210X297), and the company ’s 4 specifications (210X482) Description of the Invention (142) ATAAACAAAA GAGTTTGTAG AAACGCAAAA AGGCCATCCG TCAGGATGGC CTTCTGCTTA 1320 ATTTGATGCC TGGCAGTTTA TGGCGGGCGT CCTGCCCGCC ACCCTCCGGG CCGTTGCTTC 1380 GCAACGTTC A AATCCGCTCC CGGCGGATTT GTCCTACTCA GGAGAGCGTT CACCGACAAA 1440 CAACAGATAA AACGAAAGGC CCAGTCTTTC GACTGAGCCT TTCGTTTTAT TTGATGCCTG 1500 GCAGTTCCCT ACTCTCGCAT GGGGAGACCA TGCATAC 1537 (2) SEQ ID NO · 47 Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明·· SEQ ID NO : 47 : CCGGCGGACA TTTATCACAC AGCAGCTGAT GAGAAGTTTC TTCATCCA 48 (2) SEQ ID NO : 48的資訊： (i) 序列特性： (A) 長度·· 55鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description. SEQ ID NO: 47: CCGGCGGACA TTTATCACAC AGCAGCTGAT GAGAAGTTTC TTCATCCA 48 (2) Information of SEQ ID NO: 48: (i) Sequence characteristics: (A) Length · 55 Alkali pair (B) Category: Nucleic acid (C) Strand type ·· Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO ·· 48 ·· OGATTIGATT CTAGAAGGAG GAATAACATA TGGFITAACGC GITGGAATrC GGTAC 55 (2) SEQ ID NO : 49的資訊： (i) 序列特性： (A) 長度·· 49鹼對 (B) 類別：核酸 (C) 股型。·單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO 48 48 OGATTIGATT CTAGAAGGAG GAATAACATA TGGFITAACGC GITGGAATrC GGTAC 55 (2) Information of SEQ ID NO: 49: (i) Sequence characteristics: (A) Length · 49 base pairs (B) category: nucleic acid (C) strand type. · Single strand (D) Topology: Linear

(ii) 分子類別：CDNA -145- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） ---------— (請先閱讀背面之注意事項再填寫本頁) 訂，線 2 8 4 2 2 經濟部中央標準局員工消費合作社印製 A7 ___B7 五、發明説明（143) (xi)序列説明：SEQ ID NO : 49 : CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49 (2) SEQ ID NO : 50的資訊： (i) 序列特性： (A) 長度：1546鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: CDNA -145- This paper size is applicable to Chinese National Standard (CNS) A4 specification (210 × 297 mm) ---------— (Please read the precautions on the back before filling this page) Order, line 2 8 4 2 2 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 ___B7 V. Description of invention (143) (xi) Sequence description: SEQ ID NO: 49: CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49 (2) SEQ ID Information for NO: 50: (i) Sequence characteristics: (A) Length: 1546 Base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明·· SEQ ID NO ·· 50 ·· GCGTAACGTA TGCATGGTCT CCCCATGCGA GAGTAGGGAA CTGCCAGGCA TCAAATAAAA 60 CGAAAGGCTC AGTCGAAAGA CTGGGCCTTT CGTTTTATCT GTTGTTTGTC GGTGAACGCT 120 CTCCTGAGTA GGACAAATCC GCCGGGAGCG GATTTGAACG TTGCGAAGCA ACGGCCCGGA 180 GGGTGGCGGG CAGGACGCCC GCCATAAACT GCCAGGCATC AAATTAAGCA GAAGGCCATC 240 CTGACGGATG GCCTTTTTGC GTTTCTACAA ACTCTTTTGT TTATTTTTCT AAATACATTC 300 AAATATGGAC GTCGTACTTA ACTTTTAAAG TATGGGCAAT CAATTGCTCC TGTTAAAATT 360 GCTTTAGAAA TACTTTGGCA GCGGTTTGTT GTATTGAGTT TCATTTGCGC ATTGGTTAAA 420 TGGAAAGTGA CCGTGCGCTT ACTACAGCCT AATATTTTTG AAATATCCCA AGAGCTTTTT 480 CCTTCGCATG CCCACGCTAA ACATTCTTTT TCTCTTTTGG TTAAATCGTT GTTTGATTTA 540 TTATTTGCTA TATTTATTTT TCGATAATTA TCAACTAGAG AAGGAACAAT TAATGGTATG 600 TTCATACACG CATGTAAAAA TAAACTATCT ATATAGTTGT CTTTCTCTGA ATGTGCAAAA 660 CTAAGCATTC CGAAGCCATT ATTAGCAGTA TGAATAGGGA AACTAAACCC AGTGATAAGA 720 CCTGATGATT TCGCTTCTTT AATTACATTT GGAGATTTTT TATTTACAGC ATTGTTTTCA 780 AATATATTCC AATTAATCGG TGAATGATTG GAGTTAGAAT AATCTACTAT AGGATCATAT 840 TTTATTAAAT TAGCGTCATC ATAATATTGC CTCCATTTTT TAGGGTAATT ATCCAGAATT 900 GAAATATCAG ATTTAACCAT AGAATGAGGA TAAATGATCG CGAGTAAATA ATATTCACAA 960 TGTACCATTT TAGTCATATC AGATAAGCAT TGATTAATAT CATTATTGCT TCTACAGGCT 1020 TTAATTTTAT TAATTATTCT GTAAGTGTCG TCGGCATTTA TGTCTTTCAT ACCCATCTCT 1080 -146- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 裝· 訂 1221482 A7 B7 五、發明説明（144) n - 1 經濟部中央標準局員工消費合作社印製 TTATCCTTAC CTATTGTTTG TCGCAAGTTT TGCGTGTTAT ATATCATTAA AACGGTAATA 1140 GATTGACATT TGATTCTAAT AAATTGGATT TTTGTCACAC TATTATATCG CTTGAAATAC 1200 AATTGTTTAA CATAAGTACC TGTAGGATCQ TACAGGTTTA CGCAAGAAAA TGGTTTGTTA 1260 TAGTCGATTA ATCGATTTGA TTCTAGATTT GTTTTAACTA ATTAAAGGAG GAATAACATA 1320 TGGTTAACGC GTTGGAATTC GAGCTCACTA GTGTCGACCT GCAGGGTACC ATGGAAGCTT 1380 ACTCGAGGAT CCGCGGAAAG AAGAAGAAGA AGAAGAAAGC CCGAAAGGAA GCTGAGTTGG 1440 CTGCTGCCAC CGCTGAGCAA TAACTAGCAT AACCCCTTGG GGCCTCTAAA CGGGTCTTGA 1500 GGGGTTTTTT GCTGAAAGGA GGAACCGCTC TTCACGCTCT TCACGC 1546 (2) SEQ ID NO : 51的資訊： ⑴序列特性： (A) 長度：47鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型(ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 51 :TATGAAACAT CATCACCATC ACCATCATGC TAGCGTTAAC GCCjTTGG 47 (2) SEQ ID NO : 52的資訊： (i) 序列特性： (A) 長度：49鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 52 :AATTCCAACG CGTTAACGCT AGCATGATGG TGATGGTGAT GATGTTTCA 49 (請先閲讀背面之注意事項再填寫本頁) 裝· 訂 -147- 本紙張尺度適用中國國家標準（CNS ) A4規格（2!0X297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（145) (2) SEQ ID NO : 53的資訊·· (i) 序列特性·· (A) 長度：141鹼對 (B) 類別··核酸 (C) 股型：單股 (D) 拓樸學：線型(Ii) Molecular Type: cDNA (xi) Sequence Description ·· SEQ ID NO ·· 50 ·· GCGTAACGTA TGCATGGTCT CCCCATGCGA GAGTAGGGAA CTGCCAGGCA TCAAATAAAA 60 CGAAAGGCTC AGTCGAAAGA CTGGGCCTTT CGTTTTATCT GTTGTTTGTC GGTGAACGCT 120 CTCCTGAGTA GGACAAATCC GCCGGGAGCG GATTTGAACG TTGCGAAGCA ACGGCCCGGA 180 GGGTGGCGGG CAGGACGCCC GCCATAAACT GCCAGGCATC AAATTAAGCA GAAGGCCATC 240 CTGACGGATG GCCTTTTTGC GTTTCTACAA ACTCTTTTGT TTATTTTTCT AAATACATTC 300 AAATATGGAC GTCGTACTTA ACTTTTAAAG TATGGGCAAT CAATTGCTCC TGTTAAAATT 360 GCTTTAGAAA TACTTTGGCA GCGGTTTGTT GTATTGAGTT TCATTTGCGC ATTGGTTAAA 420 TGGAAAGTGA CCGTGCGCTT ACTACAGCCT AATATTTTTG AAATATCCCA AGAGCTTTTT 480 CCTTCGCATG CCCACGCTAA ACATTCTTTT TCTCTTTTGG TTAAATCGTT GTTTGATTTA 540 TTATTTGCTA TATTTATTTT TCGATAATTA TCAACTAGAG AAGGAACAAT TAATGGTATG 600 TTCATACACG CATGTAAAAA TAAACTATCT ATATAGTTGT CTTTCTCTGA ATGTGCAAAA 660 CTAAGCATTC CGAAGCCATT ATTAGCAGTA TGAATAGGGA AACTAAACCC AGTGATAAGA 720 CCTGATGATT TCGCTTCTTT AATTACATTT GGAGATTTTT TATTTACAGC ATTGTTTTCA 780 AATATATTC C AATTAATCGG TGAATGATTG GAGTTAGAAT AATCTACTAT AGGATCATAT 840 TTTATTAAAT TAGCGTCATC ATAATATTGC CTCCATTTTT TAGGGTAATT ATCCAGAATT 900 GAAATATCAG ATTTAACCAT AGAATGAGGA TAAATGATCG CGAGTAAATA ATATTCACAA 960 TGTACCATTT TAGTCATATC AGATAAGCAT TGATTAATAT CATTATTGCT TCTACAGGCT 1020 TTAATTTTAT TAATTATTCT GTAAGTGTCG TCGGCATTTA TGTCTTTCAT ACCCATCTCT 1080 -146- This paper scales applicable Chinese National Standard (CNS) A4 size (210X297 mm) (Please read the precautions on the back before filling out this page) Binding · Order 1221482 A7 B7 V. Invention Description (144) n-1 Printed by the Consumer Cooperatives of the Central Standards Bureau, Ministry of Economic Affairs TTATCCTTAC CTATTGTTTG TCGCAAGTTT TGCGTGTTAT ATATCATTAA AACGGTAATA 1140 GATTGACATT TGATTCTAAT AAATTGGATT TTTGTCACAC TATTATATCG CTTGAAATAC 1200 AATTGTTTAA CATAAGTACC TGTAGGATCQ TACAGGTTTA CGCAAGAAAA TGGTTTGTTA 1260 TAGTCGATTA ATCGATTTGA TTCTAGATTT GTTTTAACTA ATTAAAGGAG GAATAACATA 1320 TGGTTAACGC GTTGGAATTC GAGCTCACTA GTGTCGACCT GCAGGGTACC ATGGAAGCTT 1380 ACTCGAGGAT CCGCGGAAAG AAGAAGAAGA AGAAGAAAGC CCGAAAGGAA GCTGAGTTGG 1440 CTGCTGCCAC CGCTGAGCAA TAACTAGCAT AACCCCTTGG GGCCTCTAAA CGGGTCTTGA 1500 GGGGTTTTTT GCTGAAAGGA GGAACCGCTC TTCACGCTCT TCACGC 1546 (2) Information of SEQ ID NO: 51: ⑴ Sequence characteristics: (A) Length: 47 base pairs (B) · Single strand (D) topology: linear (ii) molecular category: cDNA (xi) sequence description: SEQ ID NO: 51: TATGAAACAT CATCACCATC ACCATCATGC TAGCGTTAAC GCCjTTGG 47 (2) information of SEQ ID NO: 52: (i) sequence Characteristics: (A) Length: 49 base pairs (B) Category: Nucleic acid (C) Strand type ·· Single strand (D) Topology: Linear type (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 52 : AATTCCAACG CGTTAACGCT AGCATGATGG TGATGGTGAT GATGTTTCA 49 (Please read the precautions on the back before filling out this page) Binding · Order -147- This paper size applies to the Chinese National Standard (CNS) A4 specification (2! 0X297 mm) 1221482 Central Standard of the Ministry of Economic Affairs Printed by the Consumer Cooperatives of the Bureau A7 B7 V. Description of the invention (145) (2) Information of SEQ ID NO: 53 (i) Sequence characteristics ... (A) Length: 141 base pairs (B) Category ... Nucleic acid ( C) Stock type: single stock ( D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 53 : CTAATTCCGC TCTCACCTAC CAAACAATGC CCCCCTGCAA AAAATAAATT CATATAAAAA 60 ACATACAGAT AACCATCTGC GGTGATAAAT TATCTCTGGC GGTGTTGACA TAAATACCAC 120 TGGCGGTGAT ACTGAGCACA T 141 (2) SEQ ID NO : 54的資訊： (i) 序列特性： (A) 長度：147鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 53: CTAATTCCGC TCTCACCTAC CAAACAATGC CCCCCTGCAA AAAATAAATT CATATAAAAA 60 ACATACAGAT AACCATCTGC GGTGATAAAT TATCTCTGGC GGTGTTGACA TAAATACCAC 120 TGGCGGTGAT ACTGAGCACA ID ) Sequence characteristics: (A) Length: 147 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear type

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 54 : CGATGTGCTC AGTATCACCG CCAGTGGTAT TTATGTCAAC ACCGCCAGAG ATAATTTATC 60 ACCGCAGATG GTTATCTGTA TGTTTTTTAT ATGAATTTAT TTTTTGCAGG GGGGCATTGT 120 TTGGTAGGTG AGAGCGGAAT TAGACGT 147 (2) SEQ ID NO : 55的資訊： (i) 序列特性： (A) 長度：55鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 54: CGATGTGCTC AGTATCACCG CCAGTGGTAT TTATGTCAAC ACCGCCAGAG ATAATTTATC 60 ACCGCAGATG GTTATCTGTA TGTTTTTTAT ATGAATTTAT TTTTTGCAGG GGGGCATTGT 120 TTGGTAGGTAG SEQACGTGAIDGAAGGAGAAT ) Sequence characteristics: (A) Length: 55 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear type

(ii) 分子類別·· cDNA (xi)序列説明·· SEQ ID NO ·· 55 : -148- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 裝·(ii) Molecular type · cDNA (xi) sequence description · SEQ ID NO · 55: -148- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back first (Fill in this page again)

、1T 1221482 A7 B7 五、發明説明（146) CGATITGATT CTAGAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC GGTAC 55 (2) SEQ ID NO : 56的資訊： (i) 序列特性： (A) 長度：49鹼對 (B) 類別··核酸 (C) 股型：單股 (D) 拓樸學：線型1T 1221482 A7 B7 V. Description of the invention (146) CGATITGATT CTAGAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC GGTAC 55 (2) Information of SEQ ID NO: 56: (i) Sequence characteristics: (A) Length: 49 base pairs (B) Category ·· Nucleic Acid (C) Strand: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 56 : CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49 (2) SEQ ID NO : 57的資訊： (i) 序列特性： (A) 長度·· 668鹼對 (B) 類別··核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 56: CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49 (2) Information of SEQ ID NO: 57: (i) Sequence characteristics: (A) Length · 668 base Pair (B) Category · Nucleic Acid (C) Strand: Single Strand (D) Topology: Linear

(ii) 分子類別·· cDNA (xi)序列説明：SEQ ID NO : 57 : (請先閲讀背面之注意事項再填寫本頁) •裝· 訂經濟部中央標準局員工消費合作衽印製 GTGAAGAGCG TGAAGAGCGG TTCCTCCTTT CAGCAAAAAA CCCCTCAAGA CCCGTTTAGA 60 GGCCCCAAGG GGTTATGCTA GTTATTGCTC AGCGGTGGCA GCAGCCAACT CAGGTTCCTT 120 TCGGGCTTTC TTCTTCTTCT TCTTCTTTCC GCGGATCCTC GAGTAAGCTT CCATGGTACC 180 CTGCAGGTCG ACACTAGTGA GCTCGAATTC CAACGCGTTA ACCATATGTT ATTCCTCCTT 240 TAATTAGTTA ACTCAAATCT AGAATCAAAT CGATAAATTG TGAGCGCTCA CAATTGAGAA 300 TATTAATCAA GAATTTTAGC ATTTGTCAAA TGAATTTTTT AAAAATTATG AGACGTCCAT 360 ATTTGAATGT ATTTAGAAAA ATAAACAAAA GAGTTTGTAG AAACGCAAAA AGGCCATCCG 420 TCAGGATGGC CTTCTGCTTA ATTTGATGCC TGGCAGTTTA TGGCGGGCGT CCTGCCCGCC 480 ACCCTCCGGG CCGTTGCTTC GCAACGTTCA AATGCGCTCC CGGCGGATTT GTCCTACTCA 540 GGAGAGCGTT CACCGACAAA CAACAGATAA AACGAAAGGC CCAGTCTTTC GACTGAGCCT 600 149 1221482 A7 _B7____ 五、發明説明（147) TTCGTTTTAT TTGATGCCTG GCAGTTCCCT ACTCTCGCAT GGGGAGACCA TGCATACGTT 660 ACGCACGT 668 (2) SEQ ID NO ·· 58的資訊·· (i) 序列特性： (A) 長度·· 726鹼對 (B) 類別··核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) Molecular type · · cDNA (xi) sequence description: SEQ ID NO: 57: (Please read the notes on the back before filling out this page) • Binding · Ordering the consumption cooperation of the Central Standards Bureau of the Ministry of Economic Affairs 衽 Printing GTGAAGAGCG TGAAGAGCGG TTCCTCCTTT CAGCAAAAAA CCCCTCAAGA CCCGTTTAGA 60 GGCCCCAAGG GGTTATGCTA GTTATTGCTC AGCGGTGGCA GCAGCCAACT CAGGTTCCTT 120 TCGGGCTTTC TTCTTCTTCT TCTTCTTTCC GCGGATCCTC GAGTAAGCTT CCATGGTACC 180 CTGCAGGTCG ACACTAGTGA GCTCGAATTC CAACGCGTTA ACCATATGTT ATTCCTCCTT 240 TAATTAGTTA ACTCAAATCT AGAATCAAAT CGATAAATTG TGAGCGCTCA CAATTGAGAA 300 TATTAATCAA GAATTTTAGC ATTTGTCAAA TGAATTTTTT AAAAATTATG AGACGTCCAT 360 ATTTGAATGT ATTTAGAAAA ATAAACAAAA GAGTTTGTAG AAACGCAAAA AGGCCATCCG 420 TCAGGATGGC CTTCTGCTTA ATTTGATGCC TGGCAGTTTA TGGCGGGCGT CCTGCCCGCC 480 ACCCTCCGGG CCGTTGCTTC GCAACGTTCA AATGCGCTCC CGGCGGATTT GTCCTACTCA 540 GGAGAGCGTT CACCGACAAA CAACAGATAA AACGAAAGGC CCAGTCCTTCGCTCGCTCTCGC 149 CGTACTCTCT_GC7 T 660 ACGCACGT 668 (2) Information of SEQ ID NO · 58 · (i) sequence characteristics: (A) length · 726 base pairs (B) category · nucleic acid (C) strand type · single strand (D ) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO ·· 58 : (請先閱讀背面之注意事項再填寫本頁) 裝. 、?τ 經濟部中央榡準局員工消費合作、社印製 GCGTAACGTA TGCATGGTCT CCCCATGCGA GAGTAGGGAA CTGCCAGGCA TCAAATAAAA 60 CGAAAGGCTC AGTCGAAAGA CTGGGCCTTT CGTTTTATCT GTTGTTTGTC GGTGAACGCT 120 CTCCTGAGTA GGACAAATCC GCCGGGAGCG GATTTGAACG TTGCGAAGCA ACGGCCCGGA 180 GGGTGGCGGG CAGGACGCCC GCCATAAACT GCCAGGCATC AAATTAAGCA GAAGGGGCCT 240 CCCACCGCCC GTCCTGCGGG CGGTATTTGA CGGTCCGTAG TTTAATTCGT CTTCGCCATC 300 CTGACGGATG GCCTTTTTGC GTTTCTACAA ACTCTTTTGT TTATTTTTCT AAATACATTC 360 AAATATGGAC GTCTCATAAT TTTTAAAAAA TTCATTTGAC AAATGCTAAA ATTCTTGATT 420 AATATTCTCA ATTGTGAGCG CTCACAATTT ATCGATTTGA TTCTAGATTT GTTTTAACTA 480 ATTAAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC GAGCTCACTA GTGTCGACCT 540 GCAGGGTACC ATGGAAGCTT ACTCGAGGAT CCGCGGAAAG AAGAAGAAGA AGAAGAAAGC 600 CCGAAAGGAA GCTGAGTTGG CTGCTGCCAC CGCTGAGCAA TAACTAGCAT AACCCCTTGG 660 GGCCTCTAAA CGGGTCTTGA GGGGTTTTTT GCTGAAAGGA GGAACCGCTC TTCACGCTCT 720 TCACGC 726 -150- 本紙張尺度適用中國國家標準（CNS ) A4規格（2l〇X297公釐） 1221482 A7 B7 五、發明説明（148) (2) SEQ ID NO : 59的資訊： (i) 序列特性： (A) 長度：44鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO · · 58: (Please read the precautions on the back before filling out this page). prepared GCGTAACGTA TGCATGGTCT CCCCATGCGA GAGTAGGGAA CTGCCAGGCA TCAAATAAAA 60 CGAAAGGCTC AGTCGAAAGA CTGGGCCTTT CGTTTTATCT GTTGTTTGTC GGTGAACGCT 120 CTCCTGAGTA GGACAAATCC GCCGGGAGCG GATTTGAACG TTGCGAAGCA ACGGCCCGGA 180 GGGTGGCGGG CAGGACGCCC GCCATAAACT GCCAGGCATC AAATTAAGCA GAAGGGGCCT 240 CCCACCGCCC GTCCTGCGGG CGGTATTTGA CGGTCCGTAG TTTAATTCGT CTTCGCCATC 300 CTGACGGATG GCCTTTTTGC GTTTCTACAA ACTCTTTTGT TTATTTTTCT AAATACATTC 360 AAATATGGAC GTCTCATAAT TTTTAAAAAA TTCATTTGAC AAATGCTAAA ATTCTTGATT 420 AATATTCTCA ATTGTGAGCG CTCACAATTT ATCGATTTGA TTCTAGATTT GTTTTAACTA 480 ATTAAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC GAGCTCACTA GTGTCGACCT 540 GCAGGGTACC ATGGAAGCTT ACTCGAGGAT CCGCGGAAAG AAGAAGAAGA AGAAGAAAGC 600 CCGAAAGGAA GCTGAGTTGG CTGCTGCCAC CGCTGAGCAA TAACTAGCAT AACCCCTTGG 660 GGCCTCTAAA CGGGTCTTGA GGGGTTTTTT GCTGAAAGGA GGAAC CGCTC TTCACGCTCT 720 TCACGC 726 -150- This paper size applies to Chinese National Standard (CNS) A4 (2l0 × 297mm) 1221482 A7 B7 V. Description of the invention (148) (2) Information of SEQ ID NO: 59: (i ) Sequence characteristics: (A) Length: 44 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear type

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 59 : TACGACTGG ATCCTTATAA GCAGCTTATT TTTACTGATT GGAC 44 (2) SEQ ID NO : 60的資訊： (i) 序列特性： (A) 長度：27鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 59: TACGACTGG ATCCTTATAA GCAGCTTATT TTTACTGATT GGAC 44 (2) Information of SEQ ID NO: 60: (i) Sequence characteristics: (A) Length: 27 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 60 : GTCCTCCTGG TACCTACCTA AAACAAC 27 (2) SEQ ID NO : 61 的資訊： (i) 序列特性： (A) 長度：102鹼對 (B) 類別··核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 60: GTCCTCCTGG TACCTACCTA AAACAAC 27 (2) Information of SEQ ID NO: 61: (i) Sequence characteristics: (A) Length: 102 base pairs (B ) Type ·· Nucleic Acid (C) Strand: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) (xi)序列説明：SEQ ID NO : 61 : TATGGATGAA GAAACTTCTC ATCAGCTGCT GTGTGATAAA TGTCCGCCGG GTACCCGGCG 60 GACATTTATC ACACAGCAGC TGATGAGAAG TTTCTTCATC CA 102 (2) SEQ ID NO : 62的資訊： (i)序列特性： (A)長度：19胺基酸 -151 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐) 1221482 A7 ____B7 五、發明説明（149) (B) 類別：胺基酸 (C) 股型：單股 (D) 拓樸學：線型 (ii)分子類別：蛋白質 (xi)序列説明：SEQ ID NO : 62 :(ii) Molecular type: cDNA printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) (xi) Sequence description: SEQ ID NO: 61: TATGGATGAA GAAACTTCTC ATCAGCTGCT GTGTGATAAA TGTCCGCCGG GTACCCGGCG 60 GACATTTATC ACACAGCAGC TGATGAGAAG TTTCTTCATC CA 102 (2) Information of SEQ ID NO: 62: (i) Sequence characteristics: (A) Length: 19 amino acid-151-This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) ) 1221482 A7 ____B7 V. Description of the invention (149) (B) Category: Amino acid (C) Strand type: Single strand (D) Topology: Linear (ii) Molecular category: Protein (xi) Sequence description: SEQ ID NO : 62:

Met Asp Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro 15 10 15Met Asp Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro 15 10 15

Gly Thr Tyr (2) SEQ ID NO : 63的資訊： (i) 序列特性： (A) 長度：84鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型Gly Thr Tyr (2) Information of SEQ ID NO: 63: (i) Sequence characteristics: (A) Length: 84 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO ·· 63 : TATGGAAACT TTTCCTCCAA AATATCTTCA TTATGATGAA GAAACTTCTC ATCAGCTGCT 60 GTGTGATAAA TGTCCGCCGG GTAC 84 (2) SEQ ID NO : 64的資訊： (i) 序列特性： (A) 長度·· 78鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁)(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO · 63: TATGGAAACT TTTCCTCCAA AATATCTTCA TTATGATGAA GAAACTTCTC ATCAGCTGCT 60 GTGTGATAAA TGTCCGCCGG GTAC 84 (2) Information of SEQ ID NO: 64: (i) Sequence characteristics: (A ) Length ·· 78 base pairs (B) Category: Nucleic acid (C) Stock type: Single strand (D) Topology: Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Linear Economy (Please read the notes on the back before filling out this page )

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 64 : CCGGCGGACA TTTATCACAC AGCAGCTGAT GAGAAGTTTC TTCATCATAA TGAAGATATT 60 TTGGAGGAAA AGTTTCCA 78 (2) SEQ ID NO : 65的資訊·· (i)序列特性： •152- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X29?公釐） 1221482 A 7 B7 五、發明説明（150) 經濟部中央標準局員工消費合作衽印製 (A) 長度：44鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 65 : TACGCACTGG ATCCTTATAA GCAGCTTATT TTCACGGATT GAAC 44 (2) SEQ ID NO : 66的資訊： ⑴序列特性： (A) 長度：3 8驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 66 : GTGCTCCTGG TACCTACCTA AAACAGCACT GCACAGTG 38 (2) SEQ ID NO : 67的資訊： (i) 序列特性： (A) .長度：84鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 67 : TATGGAAACT CTGCCTCCAA AATACCTGCA TTACGATCCG GAAACTGGTC ATCAGCTGCT 60 GTGTGATAAA TGTGCTCCGG GTAC 84 (2) SEQ ID NO : 68的資訊： (i)序列特性： (A) 長度：78鹼對 (B) 類別：核酸 (C) 股型··單股 (請先閲讀背面之注意事項再填寫本頁) 裝· 、?τ -153- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 經濟部中央標準局員工消費合作衽印製 A7 B7___ 五、發明説明（151) (D)拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 68 : CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC CGGATCGTAA TGCAGGTATT 60 TTGGAGGCAG AGTTTCCA 78 (2) SEQ ID NO : 69的資訊： (i) 序列特性： (A) 長度：54鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 64: CCGGCGGACA TTTATCACAC AGCAGCTGAT GAGAAGTTTC TTCATCATAA TGAAGATATT 60 TTGGAGGAAA AGTTTCCA 78 (2) Information of SEQ ID NO: 65 · (i) Sequence characteristics: • 152- This paper size applies Chinese National Standard (CNS) A4 (210X29? Mm) 1221482 A 7 B7 V. Description of invention (150) Consumption cooperation printed by employees of the Central Standards Bureau of the Ministry of Economic Affairs (A) Length: 44 alkali pairs (B ) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear type (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 65: TACGCACTGG ATCCTTATAA GCAGCTTATT TTCACGGATT GAAC 44 (2) SEQ ID NO : 66 Information: ⑴ Sequence characteristics: (A) Length: 3 8 pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear (ii) Molecular type: cDNA (xi) sequence Description: SEQ ID NO: 66: GTGCTCCTGG TACCTACCTA AAACAGCACT GCACAGTG 38 (2) Information of SEQ ID NO: 67: (i) Sequence characteristics: (A). Length: 84 base pairs (B) Category: Nucleic acid (C) Strand type : Single strand (D) topology: linear (ii) molecular class: cDNA (xi) sequence description: SEQ ID NO: 67: TATGGAAACT CTGCCTCCAA AATACCTGCA TTACGATCCG GAAACTGGTC ATCAGCTGCT 60 GTGTGATAAA TGTGCTCCGG GTAC 84 (2) Information of SEQ ID NO: 68: (i) Sequence characteristics: (A) Length: 78 base pairs (B) Category: nucleic acid (C ) Stock type ·· Single stock (please read the precautions on the back before filling this page). ·· τ -153- This paper size is applicable to the Chinese National Standard (CNS) A4 size (210X297 mm) 1221482 Central Standard of the Ministry of Economic Affairs A7 B7___ printed by the Bureau ’s consumer cooperation V. Description of the invention (151) (D) Topology: Linear (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 68: CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC CGGATCGTAA TGCAGGTATT 60 TTGGAGGCAG AGTTTCCA 78 (2) Information of SEQ ID NO: 69: (i) Sequence characteristics: (A) Length: 54 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 69 : TATGGACCCA GAAACTGGTC ATCAGCTGCT GTGTGATAAA TGTGCTCCGG GTAC 54 (2) SEQ ID NO : 70的資訊： (i) 序列特性： (A) 長度：48鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 69: TATGGACCCA GAAACTGGTC ATCAGCTGCT GTGTGATAAA TGTGCTCCGG GTAC 54 (2) Information of SEQ ID NO: 70: (i) Sequence characteristics: (A) Length: 48 bases Pair (B) Category: Nucleic Acid (C) Strand Type · Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 70 : CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC TGGGTCCA 48 (2) SEQ ID NO : 71的資訊： (i) 序列特性： (A) 長度：87鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 70: CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC TGGGTCCA 48 (2) Information of SEQ ID NO: 71: (i) Sequence characteristics: (A) Length: 87 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 71 : _____ -154- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁) -裝· 訂 1221482 A7 B7 五、發明説明（152) 經濟部中央標準局員工消費合作社印繁(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 71: _____ -154- This paper size is applicable to China National Standard (CNS) A4 (210X 297 mm) (Please read the precautions on the back before (Fill in this page)-Binding and ordering 1221482 A7 B7 V. Description of invention (152) Employees' Cooperatives of Central Standards Bureau of Ministry of Economic Affairs

TATGAAAGAA ACTCTGCCTC CAAAATACCT GCATTACGAT CCGGAAACTG GTCATCAGCT GCTGTGTGAT AAATGTGCTC CGGGTAC(2) SEQ ID NO : 72的資訊：(i) 序列特性：(A) 長度：81鹼對(B) 類別：核酸(C) 股型：單股(D) 拓樸學：線型(ii) 分子類別：cDNA(xi)序列説明：SEQ ID NO : 72 : CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC CGGATCGTAA TGCAGGTATT TTGGAGGCAG AGTTTCTTTC A(2) SEQ ID NO : 73的資訊：(i) 序列特性：(A) 長度：7 1鹼對(B) 類別··核酸(C) 股型：單股(D) 拓樸學：線型(ii) 分子類別：cDNA(xi)序列説明：SEQ ID NO : 73 : GTTCTCCTCA TATGAAACAT CATCACCATC ACCATCATGA AACTCTGCCT CCAAAATACC TGCATTACGA T(2) SEQ ID NO : 74的資訊：(i) 序列特性(A) 長度(B) 類別(C) 股型(D) 拓樸學：線型(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO 60 87 60 81 60 71 (請先閲讀背面之注意事項再填寫本頁) -裝· 43鹼對核酸單股 74 -155 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（153) CTTCTCCTCA TATGAAAGAA ACTCTGCCTC CAAAATACCT GCA 43 (2) SEQ ID NO : 75的資訊： (i) 序列特性： (A) 長度：76鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型TATGAAAGAA ACTCTGCCTC CAAAATACCT GCATTACGAT CCGGAAACTG GTCATCAGCT GCTGTGTGAT AAATGTGCTC CGGGTAC (2) Information of SEQ ID NO: 72: (i) Sequence characteristics: (A) Length: 81 base pairs (B) Category: Nucleic acid (C) Share type: Single strand (D ) Topology: Linear (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 72: CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC CGGATCGTAA TGCAGGTATT TTGGAGGCAG AGTTTCTTTC A (2) Information of SEQ ID NO: 73: (i) Sequence characteristics: (A) Length: 71 Base pairs (B) Category · Nucleic acid (C) Strand type: Single strand (D) Topology: Linear type (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 73: GTTCTCCTCA TATGAAACAT CATCACCATC ACCATCATGA AACTCTGCCT CCAAAATACC TGCATTACGA T (2) Information of SEQ ID NO: 74: (i) sequence characteristics (A) length (B) category (C) share type (D) topology: linear type (ii) molecular type: cDNA (xi) sequence description: SEQ ID NO 60 87 60 81 60 71 (please read the precautions on the back before filling this page)-installed · 43 alkali to nucleic acid single strand 74 -155 This paper size applies Chinese national standard (CNS ) A4 specifications (210X297 mm) 1221482 A7 B7 Description (153) CTTCTCCTCA TATGAAAGAA ACTCTGCCTC CAAAATACCT GCA 43 (2) Information of SEQ ID NO: 75: (i) Sequence characteristics: (A) Length: 76 base pairs (B) Category: Nucleic acid (C) Genotype ·· single (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 75 : TACGCACTGG ATCCTTAATG ATGGTGATGG TGATGATGTA AGCAGCTTAT TTTCACGGAT 60 TGAACCTGAT TCCCTA 7 6 (2) SEQ ID NO : 76的資訊： (i) 序列特性： (A) 長度：47鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 75: TACGCACTGG ATCCTTAATG ATGGTGATGG TGATGATGTA AGCAGCTTAT TTTCACGGAT 60 TGAACCTGAT TCCCTA 7 6 (2) Information of SEQ ID NO: 76: (i) Sequence characteristics: (A) Length: 47 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 76 : GTTCTCCTCA TATGAAATAC CTGCATTACG ATCCGGAAAC TGGTCAT 47 (2) SEQ ID NO : 77的資訊： ⑴序列特性： (A) 長度：43鹼對 (B) 類別：核酸經濟部中央標準局員工消費合作衽印製 (請先閲讀背面之注意事項再填寫本頁) (C) 股型：單股(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 76: GTTCTCCTCA TATGAAATAC CTGCATTACG ATCCGGAAAC TGGTCAT 47 (2) Information of SEQ ID NO: 77: ⑴ Sequence characteristics: (A) Length: 43 base pairs (B ) Category: Printed by the Consumer Standards Cooperative Standards of the Central Standards Bureau of the Ministry of Nucleic Acid Economy (Please read the precautions on the back before filling out this page) (C) Stock type: single stock

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 77 : GTTCTCCTAT TAATGAAATA TCTTCATTAT GATGAAGAAA CTT 43 (2) SEQ ID NO : 78的資訊： (i)序列特性： -156- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇'〆297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 _ 五、發明説明（154) (A) 長度：40鹼對 (B) 類別：核酸 (C) 股型：單股(D) Topology: Linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 77: GTTCTCCTAT TAATGAAATA TCTTCATTAT GATGAAGAAA CTT 43 (2) Information of SEQ ID NO: 78: (i) Sequence characteristics:- 156- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (21〇'297 mm) 1221482 Printed by the Consumers' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 _ 5. Description of the invention (154) (A) Length: 40 Alkali pair (B) Category: Nucleic acid (C) Share type: Single strand

(D) 拓樸學：線型 (ii)分子類別cDNA (xi)序列説明：SEQ ID NO : 78 : TACGCACTGG ATCCTTATAA GCAGCTTATT TTTACTGATT 40 (2) SEQ ID NO : 79的資訊： (i) 序列特性： (A) 長度：40鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: linear (ii) molecular cDNA (xi) sequence description: SEQ ID NO: 78: TACGCACTGG ATCCTTATAA GCAGCTTATT TTTACTGATT 40 (2) Information of SEQ ID NO: 79: (i) Sequence characteristics: (A) Length: 40 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 79 : GTTCTCCTCA TATGGAAACT CTGCCTCCAA AATACCTGCA 40 (2) SEQ ID NO ·· 80的資訊： (i) 序列特性： (A) 長度：43鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 79: GTTCTCCTCA TATGGAAACT CTGCCTCCAA AATACCTGCA 40 (2) Information of SEQ ID NO · · 80: (i) Sequence characteristics: (A) Length: 43 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 80 : TACGCACTGG ATCCTTATGT TGCATTTCCT TTCTGAATTA GCA 43 (2) SEQ ID NO : 81的資訊： (i) 序列特性： (A) 長度：18鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 80: TACGCACTGG ATCCTTATGT TGCATTTCCT TTCTGAATTA GCA 43 (2) Information of SEQ ID NO: 81: (i) Sequence characteristics: (A) Length: 18 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA -157- 本紙張尺度適用中國國家標準（CNS ) A4規格（2丨0'〆297公釐） I ~~ 訂 (請先聞讀背面之注意事項再填寫本頁) 1221482 A7 B7 五、發明説明（1δ5) (xi)序列説明：SEQ ID NO : 81 : CCGGAAACAG ATAATGAG 18 (2) SEQ ID NO : 82的資訊： (i) 序列特性： (A) 長度：1 8驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA -157- This paper size is applicable to Chinese National Standard (CNS) A4 specification (2 丨 0'〆297mm) I ~~ Order (please read the precautions on the back before filling this page) 1221482 A7 B7 V. Description of the invention (1δ5) (xi) Sequence description: SEQ ID NO: 81: CCGGAAACAG ATAATGAG 18 (2) Information of SEQ ID NO: 82: (i) Sequence characteristics: (A) Length: 1 8 Pair (B) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 82 : GATCCTCATT ATCTGTTT 18 (2) SEQ ID NO : 83的資訊： (i) 序列特性： (A) 長度：30鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 82: GATCCTCATT ATCTGTTT 18 (2) Information of SEQ ID NO: 83: (i) Sequence characteristics: (A) Length: 30 base pairs (B) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 83 : CCGGAAACAG AGAAGCCACG CAAAAGTAAG 30 (2) SEQ ID NO : 84的資訊： (i) 序列特性： (A) 長度：30鹼對 (B) 類別：核酸 (C) 股型：單股經濟部中央標準局員工消費合作社印製 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 83: CCGGAAACAG AGAAGCCACG CAAAAGTAAG 30 (2) Information of SEQ ID NO: 84: (i) Sequence characteristics: (A) Length: 30 base pairs (B ) Category: Nucleic Acid (C) Stock Type: Single Stock Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 84 : GATCCTTACT TTTGCGTGGC TTCTCTGTTT 30 (2) SEQ ID NO : 85的資訊： (i)序列特性： -158- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（156) (A) 長度：12鹼對 (B) 類別：核酸 (C) 股型：單股(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 84: GATCCTTACT TTTGCGTGGC TTCTCTGTTT 30 (2) Information of SEQ ID NO: 85: (i) Sequence characteristics: -158- This paper size applies Chinese national standards (CNS) A4 specifications (210X297 mm) 1221482 A7 B7 V. Description of the invention (156) (A) Length: 12 base pairs (B) Category: Nucleic acid (C) Share type: Single strand

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 85 : TATGTTAATG AG 12 (2) SEQ ID NO : 86的資訊·· · (i) 序列特性： (A) 長度：14鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 85: TATGTTAATG AG 12 (2) Information of SEQ ID NO: 86. · (i) Sequence characteristics: (A ) Length: 14 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 86 : GATCCTCATT AACA 14 (2) SEQ ID NO : 87的資訊： (i) 序列特性： (A) 長度：21鹼對 (B) 類別··核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 86: GATCCTCATT AACA 14 (2) Information of SEQ ID NO: 87: (i) Sequence characteristics: (A) Length: 21 base pairs (B) Category ·· Nucleic acid (C) Strand ·· Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 87 : TATGTTCCGG AAACAGTTAA G 21 經濟部中央標準局員工消費合作社印製 (2) SEQ ID NO : 88的資訊·· (i) 序列特性： (A) 長度：2 3驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 87: TATGTTCCGG AAACAGTTAA G 21 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (2) Information of SEQ ID NO: 88. (i) Sequence characteristics : (A) Length: 2 3 Check pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA -159- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（157) (xi)序列説明：SEQ ID NO : 88 : GATCCTTAAC TGTTTCCGGA ACA 23 (2) SEQ ID NO : 89的資訊： (i) 序列特性： (A) 長度：3 6驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA -159- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 V. Description of the invention (157) (xi) Sequence description: SEQ ID NO: 88: GATCCTTAAC TGTTTCCGGA ACA 23 (2) Information of SEQ ID NO: 89: (i) Sequence characteristics: (A) Length: 3 6 Check pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 89 : TATGTTCCGG AAACAGTGAA TCAACTCAAA AATAAG 36 (2) SEQ ID NO : 90的資訊： (i) 序列特性： (A) 長度：38鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 89: TATGTTCCGG AAACAGTGAA TCAACTCAAA AATAAG 36 (2) Information of SEQ ID NO: 90: (i) Sequence characteristics: (A) Length: 38 base pairs ( B) Category: Nucleic Acid (C) Strand Type ·· Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 90 : GATCCTTATT TTTGAGTTGA TTCACTGTTT CCGGAACA 38 (2) SEQ ID NO : 91 的資訊： ⑴序列特性： (A) 長度：100鹼對 (B) 類別：核酸 (C) 股型：單股經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁)(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 90: GATCCTTATT TTTGAGTTGA TTCACTGTTT CCGGAACA 38 (2) Information of SEQ ID NO: 91: ⑴ Sequence characteristics: (A) Length: 100 base pairs (B) Category: Nucleic acid (C) Stock type: Single-share printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page)

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 91 : CTAGCGACGA CGACGACAAA GAAACTCTGC CTCCAAAATA CCTGCATTAC GATCCGGAAA 60 CTGGTCATCA GCTGCTGTGT GATAAATGTG CTCCGGGTAC 1〇〇 (2) SEQ ID NO : 92的資訊： -160- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（158) (i) 序列特性： (A) 長度：92驗對 (B) 類別··核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 91: CTAGCGACGA CGACGACAAA GAAACTCTGC CTCCAAAATA CCTGCATTAC GATCCGGAAA 60 CTGGTCATCA GCTGCTGTGT GATAAATGTG CTCCGGGTAC 1〇 (2) Information of SEQ ID NO: 92 ： -160- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 V. Description of the invention (158) (i) Sequence characteristics: (A) Length: 92 Check pairs (B) Category · · Nucleic acid (C) strand type: single strand (D) topology: linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 92 : CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC CGGATCGTAA TGCAGGTATT 60 TTGGAGGCAG AGTTTCTTTG TCGTCGTCGT CG 92 (2) SEQ ID NO : 93的資訊： (i) 序列特性： (A) 長度：26鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 92: CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC CGGATCGTAA TGCAGGTATT 60 TTGGAGGCAG AGTTTCTTTG TCGTCGTCGT CG 92 (2) Information of SEQ ID NO: 93: (i) Sequence characteristics: (A) ) Length: 26 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 93 : ACAAACACAA TCGATTTGAT ACTAGA 26 (2) SEQ ID NO : 94的資訊： (i) 序列特性： (A) 長度：50鹼對 (B) 類別：核酸 (C) 股型：單股、 (D) 拓樸學：線型、(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 93: ACAAACACAA TCGATTTGAT ACTAGA 26 (2) Information of SEQ ID NO: 94: (i) Sequence characteristics: (A) Length: 50 base pairs (B ) Category: Nucleic acid (C) Strand: Single strand, (D) Topology: Linear,

(ii) 分子類別：cDNA 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) (xi)序列説明：SEQ ID NO : 94 : TITGTnTAA CTAATTAAAG GAGGAATAAA ATATGAGAGG ATCGCATCAC 50 (2) SEQ ID NO : 95的資訊： (i)序列特性： (A) 長度：50鹼對 (B) 類別：核酸 161 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（1S9) (C) 股型：單股(ii) Molecular type: cDNA printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) (xi) Sequence description: SEQ ID NO: 94: TITGTnTAA CTAATTAAAG GAGGAATAAA ATATGAGAGG ATCGCATCAC 50 (2 ) Information of SEQ ID NO: 95: (i) Sequence characteristics: (A) Length: 50 base pairs (B) Category: Nucleic acid 161-This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 A7 B7 V. Description of the invention (1S9) (C) Stock type: single stock

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 95 : CATCACCATC ACGAAACCTT CCCGCCGAAA TACCTGCACT ACGACGAAGA 50 (2) SEQ ID NO : 96的資訊： (i) 序列特性： (A) 長度：49鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 95: CATCACCATC ACGAAACCTT CCCGCCGAAA TACCTGCACT ACGACGAAGA 50 (2) Information of SEQ ID NO: 96: (i) Sequence characteristics: (i) A) Length: 49 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 96 : AACCTCCCAC CAGCTGCTGT GCGACAAATG CCCGCCGGGT ACCCAAACA 49 (2) SEQ ID NO : 97的資訊： (i) 序列特性： (八）長度：26鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 96: AACCTCCCAC CAGCTGCTGT GCGACAAATG CCCGCCGGGT ACCCAAACA 49 (2) Information of SEQ ID NO: 97: (i) Sequence characteristics: (eight) Length: 26 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 97 : TGTTTGGGTA CCCGGCGGGC ATTTGT 26 (2) SEQ ID NO : 98的資訊： (i) 序列特性：經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) (A) 長度：50鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 97: TGTTTGGGTA CCCGGCGGGC ATTTGT 26 (2) Information of SEQ ID NO: 98: (i) Sequence characteristics: Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the notes on the back before filling this page) (A) Length: 50 alkali pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 98 : -162- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（ieo) CGCACAGCAG CTGGTGGGAG GTTTCTTCGT CGTAGTGCAG GTATTTCGGC 50 (2) SEQ ID NO : 99的資訊： ⑴序列特性： (A) 長度：49鹼對 (B) 類別：核酸 (C) 股型：單股(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 98: -162- This paper size is applicable to China National Standard (CNS) A4 specification (210X 297 mm) 1221482 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Preparation A7 B7 V. Description of the invention (ieo) CGCACAGCAG CTGGTGGGAG GTTTCTTCGT CGTAGTGCAG GTATTTCGGC 50 (2) Information of SEQ ID NO: 99: ⑴ Sequence characteristics: (A) Length: 49 base pairs (B) Category: Nucleic acid (C) Share type : Single stock

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO ·· 99 : GGGAAGGTTT CGTGATGGTG ATGGTGATGC GATCCTCTCA TATTTTATT 49 (2) SEQ ID NO : 100的資訊： (i) 序列特性： (A) 長度：50鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO · 99: GGGAAGGTTT CGTGATGGTG ATGGTGATGC GATCCTCTCA TATTTTATT 49 (2) Information of SEQ ID NO: 100: (i) Sequence characteristics: (A) Length: 50 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 100 : CCTCCTTTAA TTAGTTAAAA CAAATCTAGT ATCAAATCGA TTGTGTTTGT 50 (2) SEQ ID NO : 101的資訊： (i) 序列特性： (A) 長度：59鹼對 (B) 類別：核酸、 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 100: CCTCCTTTAA TTAGTTAAAA CAAATCTAGT ATCAAATCGA TTGTGTTTGT 50 (2) Information of SEQ ID NO: 101: (i) Sequence characteristics: (A) Length: 59 base pairs (B) Category: Nucleic acid, (C) Strand: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 101 :(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 101:

ACAAACACAA TCGATTTGAT ACTAGATTTG TTTTAACTAA TTAAAGGAGG AATAAAATG (2) SEQ ID NO : 102的資訊： (i)序列特性： (A)長度：48驗對 -163- ———— - -....... ..— n···一本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) -裝. -訂 1221482 A 7 B7 五、發明説明（161) (B) 類別：核酸 (C) 股型：單股ACAAACACAA TCGATTTGAT ACTAGATTTG TTTTAACTAA TTAAAGGAGG AATAAAATG (2) Information of SEQ ID NO: 102: (i) Sequence characteristics: (A) Length: 48 check pairs -163- ————- -....... ..— n ··· One paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling out this page)-Packing.-Order 1221482 A 7 B7 V. Description of the invention (161 ) (B) Category: Nucleic acid (C) Share type: Single strand

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 102 : CTAATTAAAG GAGGAATAAA ATGAAAGAAA CTTTTCCTCC AAAATATC 48 (2) SEQ ID NO : 103的資訊： (i) 序列特性： (A) 長度·· 31鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 102: CTAATTAAAG GAGGAATAAA ATGAAAGAAA CTTTTCCTCC AAAATATC 48 (2) Information of SEQ ID NO: 103: (i) Sequence characteristics: (i) A) Length · 31 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 103 : TGTTTGGGTA CCCGGCGGAC ATTTATCACA C 31 (2) SEQ ID NO : 104的資訊： (i) 序列特性： (A) 長度：59鹼對 (B) .類別··核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 103: TGTTTGGGTA CCCGGCGGAC ATTTATCACA C 31 (2) Information of SEQ ID NO: 104: (i) Sequence characteristics: (A) Length: 59 base pairs ( B) .Category ·· Nucleic acid (C) Strand: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 104 : ACAAACACAA TCGATTTGAT ACTAGATTTG TTTTAACTAA TTAAAGGAGG AATAAAATG 59 (2) SEQ ID NO : 105的資訊：經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) (i) 序列特性： (A) 長度·· 54鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 104: ACAAACACAA TCGATTTGAT ACTAGATTTG TTTTAACTAA TTAAAGGAGG AATAAAATG 59 (2) Information of SEQ ID NO: 105: Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please first Read the notes on the back and fill in this page) (i) Sequence characteristics: (A) Length · 54 base pairs (B) Category: Nucleic acid (C) Strand type · Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 105 : -164- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（162) CTAATTAAAG GAGGAATAAA ATGAAAAAAA AAGAAACTIT TCCTCCAAAA TATC 54 (2) SEQ ID NO : 106的資訊： (i) 序列特性： (A) 長度：31鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 105: -164- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 A7 B7 V. Description of the invention (162) CTAATTAAAG GAGGAATAAA ATGAAAAAAA AAGAAACTIT TCCTCCAAAA TATC 54 (2) Information of SEQ ID NO: 106: (i) Sequence characteristics: (A) Length: 31 base pairs (B) Category: Nucleic acid (C) Share type: Single strand (D) Topology Park Xue: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 106 : TGTTTGGGTA CCCGGCGGAC ATTTATCACA C 31 (2) SEQ ID NO : 107的資訊： (i) 序列特性： (A) 長度：44鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 106: TGTTTGGGTA CCCGGCGGAC ATTTATCACA C 31 (2) Information of SEQ ID NO: 107: (i) Sequence characteristics: (A) Length: 44 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 107 : CAGCCCGGGT AAAATGGAAA CGTTTCCTCC AAAATATCTT CATT 44 (2) SEQ ID NO : 108的資訊： (i) 序列特性： (A) 長度：44鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型經濟部中央標準局員工消費合作社印製 (請先聞讀背面之注意事項再填寫本頁)(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 107: CAGCCCGGGT AAAATGGAAA CGTTTCCTCC AAAATATCTT CATT 44 (2) Information of SEQ ID NO: 108: (i) Sequence characteristics: (A) Length: 44 base pairs (B) Type: Nucleic acid (C) Stock type: Single stock (D) Topology: Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Linear Economy (Please read the notes on the back before filling out this page)

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 108 : CGTTTCCATT TTACCCGGGC TGAGCGAGAG GCTCTTCTGC GTGT 44 (2) SEQ ID NO : 109的資訊： (i)序列特性： (A)長度：45鹼對 -165- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（163) (B) 類別：核酸 (C) 股型：單股(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 108: CGTTTCCATT TTACCCGGGC TGAGCGAGAG GCTCTTCTGC GTGT 44 (2) Information of SEQ ID NO: 109: (i) Sequence characteristics: (A) Length: 45 base pairs -165- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 V. Description of Invention (163) (B) Category: Nucleic Acid (C) Stock Type: Single Stock

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 109 : CGCTCAGCCC GGGTAAAATG GAAACGTTGC CTCCAAAATA CCTGC 45 (2) SEQ ID NO : 110的資訊： (i) 序列特性： (A) 長度：39驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 109: CGCTCAGCCC GGGTAAAATG GAAACGTTGC CTCCAAAATA CCTGC 45 (2) Information of SEQ ID NO: 110: (i) Sequence characteristics: (i) A) Length: 39 pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 110 : CCATTTTACC CGGGCTGAGC GAGAGGCTCT TCTGCGTGT 39 (2) SEQ ID NO : 111的資訊： (i) 序列特性： (A) 長度：36驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 110: CCATTTTACC CGGGCTGAGC GAGAGGCTCT TCTGCGTGT 39 (2) Information of SEQ ID NO: 111: (i) Sequence characteristics: (A) Length: 36 pairs of pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 111 : GAAAATAAGC TGCTTAGCTG CAGCTGAACC AAAATC 36 (2) SEQ ID NO : 112的資訊：經濟部中央標準局員工消費合作社印製 (i) 序列特性： (A) 長度：34鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 111: GAAAATAAGC TGCTTAGCTG CAGCTGAACC AAAATC 36 (2) Information of SEQ ID NO: 112: Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (i) Sequence characteristics : (A) Length: 34 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別·· cDNA (xi)序列説明：SEQ ID NO : 112 : -166- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（164) CAGCTGCAGC TAAGCAGCTT ATTTTCACGG ATTG 34 (2) SEQ ID NO ·· 113的資訊： (i) 序列特性： (A) 長度：36鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type · cDNA (xi) sequence description: SEQ ID NO: 112: -166- This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 Staff Consumption of Central Bureau of Standards, Ministry of Economic Affairs Printed by the cooperative V. Description of the invention (164) CAGCTGCAGC TAAGCAGCTT ATTTTCACGG ATTG 34 (2) Information of SEQ ID NO · 113: (i) Sequence characteristics: (A) Length: 36 base pairs (B) Category: Nucleic acid (C) Stock Type: Single Stock (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 113 : AAAAATAAGC TGCTTAGCTG CAGCTGAACC AAAATC 36 (2) SEQ ID NO : 114的資訊： (i) 序列特性： (A) 長度：35鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 113: AAAAATAAGC TGCTTAGCTG CAGCTGAACC AAAATC 36 (2) Information of SEQ ID NO: 114: (i) Sequence characteristics: (A) Length: 35 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明·· SEQ ID NO ·· 114 ·· CAGCTGCAGC TAAGCAGCTT ATTTTTACTG ATTGG 35 (2) SEQ ID NO : 115的資訊： (i) 序列特性： (A) 長度：102鹼對 (B) 類別：核酸、 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description. SEQ ID NO. 114 CAGCTGCAGC TAAGCAGCTT ATTTTTACTG ATTGG 35 (2) Information of SEQ ID NO: 115: (i) Sequence characteristics: (A) Length: 102 Alkali pair (B) Category: Nucleic acid, (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 115 : CTAGAAGGAG GAATAACATA TGGAAACTTT TGCTCCAAAA TATCTTCATT ATGATGAAGA 60 AACTAGTCAT CAGCTGCTGT GTGATAAATG TCCGCCGGGT AC 102 (2) SEQ ID NO : 116 的資訊·· (i)序列特性： ____ -167- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X：297公釐） (請先閱讀背面之注意事項再填寫本頁) 裝、?τ 經濟部中央標準局員工消費合作社印製 1221482 A7 __ B7 五、發明説明（165) (A) 長度：94鹼對 (B) 類別：核酸 (C) 股型：單股(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 115: CTAGAAGGAG GAATAACATA TGGAAACTTT TGCTCCAAAA TATCTTCATT ATGATGAAGA 60 AACTAGTCAT CAGCTGCTGT GTGATAAATG TCCGCCGGGT AC 102 (2) Information of SEQ ID NO: 116 · (i) Sequence characteristics: ____ -167- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X: 297 mm) (Please read the precautions on the back before filling out this page). 1221482 A7 __ B7 V. Description of the invention (165) (A) Length: 94 base pairs (B) Category: nucleic acid (C) Stock type: single strand

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 116 : CCGGCGGACA TTTATCACAC AGCAGCTGAT GACTAGTTTC TTCATCATAA TGAAGATATT 60 TTGGAGCAAA AGTTTCCATA TGTTATTCCT CCTT 94 (2) SEQ ID NO : 117的資訊： (i) 序列特性： (A) 長度：62鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 116: CCGGCGGACA TTTATCACAC AGCAGCTGAT GACTAGTTTC TTCATCATAA TGAAGATATT 60 TTGGAGCAAA AGTTTCCATA TGTTATTCCT CCTT 94 (2) Information of SEQ ID NO: 117 i) Sequence characteristics: (A) Length: 62 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 117 : CTAGAAGGAG GAATAACATA TGGAAACTTT TCCTGCTAAA TATCTTCATT ATGATGAAGA 60(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 117: CTAGAAGGAG GAATAACATA TGGAAACTTT TCCTGCTAAA TATCTTCATT ATGATGAAGA 60

AA (2) SEQ ID NO : 118的資訊： 62 (i) 序列特性： (A) 長度：62鹼對 (B) 類別：核酸 (C) 股型··單股、 (D) 拓樸學：線型AA (2) Information of SEQ ID NO: 118: 62 (i) Sequence characteristics: (A) Length: 62 base pairs (B) Category: Nucleic acid (C) Strand type · Single strand, (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 118 : CTAGTTTCTT CATCATAATG AAGATATTTA GCAGGAAAAG TTTCCATATG TTATTCCTCC 60 mm . 62 (2) SEQ ID NO : 119的資訊： (i)序列特性： (A)長度：51胺基酸 -168- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 、1 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（166) (B) 類別：胺基酸 (C) 股型：單股 (D) 拓樸學：線型 (ii)分子類別：蛋白質 (xi)序列説明：SEQ ID NO : 119 :(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 118: CTAGTTTCTT CATCATAATG AAGATATTTA GCAGGAAAAG TTTCCATATG TTATTCCTCC 60 mm. 62 (2) Information of SEQ ID NO: 119: (i) Sequence characteristics: (A) Length ： 51 amino acid-168- This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297mm) (Please read the precautions on the back before filling this page), 1 1221482 A7 B7 Staff Consumption of Central Standards Bureau, Ministry of Economic Affairs Printed by the cooperative V. Description of the invention (166) (B) Category: Amino acid (C) Strand type: Single strand (D) Topology: Linear (ii) Molecular type: Protein (xi) Sequence description: SEQ ID NO: 119:

Tyr His Tyr Tyr Asp Gin Asn Gly Arg Met Cys Glu Glu Cys His Met 1 5 10 15Tyr His Tyr Tyr Asp Gin Asn Gly Arg Met Cys Glu Glu Cys His Met 1 5 10 15

Cys Gin Pro Gly His Phe Leu Val Lys His Cys Lys Gin Pro Lys Arg 20 25 30Cys Gin Pro Gly His Phe Leu Val Lys His Cys Lys Gin Pro Lys Arg 20 25 30

Asp Thr Val Cys His Lys Pro Cys Glu Pro Gly Val Thr Tyr Thr Asp 35 40 45Asp Thr Val Cys His Lys Pro Cys Glu Pro Gly Val Thr Tyr Thr Asp 35 40 45

Asp Trp His 50 (2) SEQ ID NO : 120的資訊： (i) 序列特性： (A) 長度：2432鹼對 (B) 類別：核酸 (C) :股型：單股 (D) 拓樸學：線型 (ii) 分子類別：cDNA (ix)特徵：Asp Trp His 50 (2) Information of SEQ ID NO: 120: (i) Sequence characteristics: (A) Length: 2432 base pairs (B) Category: Nucleic acid (C): Strand type: Single strand (D) Topology: Linear (ii) Molecular class: cDNA (ix) Features:

(A) 名稱/關鍵詞：CDS (B) 位置：124···1326 (xi)序列説明：SEQ ID NO : 120 : ATCAAAGGCA GGGCATACTT CCTGTTGCCC AGACCTTATA TAAAACGTCA TGTTCGCCTG 60 GGCAGCAGAG AAGCACCTAG CACTGGCCCA GCGGCTGCCG CCTGAGGTTT CCAGAGGACC 120 ACA ATG AAC AAG TGG CTG TGC TGT GCA CTC CTG GTG TTC TTG GAC ATC 168(A) Name / Keyword: CDS (B) Position: 124 ... 1326 (xi) Sequence description: SEQ ID NO: 120: ATCAAAGGCA GGGCATACTT CCTGTTGCCC AGACCTTATA TAAAACGTCA TGTTCGCCTG 60 GGCAGCAGAG AAGCACCTAG CACTGGCCCA GCGGCTGCCG CCTGAGGTTT CCAGAGGAC A CTG TGC TGT GCA CTC CTG GTG TTC TTG GAC ATC 168

Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Phe Leu Asp lie 1 5 10 15 ATT GAA TGG ACA ACC CAG GAA ACC TTT CCT CCA AAA TAC TTG CAT TAT 216Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Phe Leu Asp lie 1 5 10 15 ATT GAA TGG ACA ACC CAG GAA ACC TTT CCT CCA AAA TAC TTG CAT TAT 216

He Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr 20 25 30 -169- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) -裝·He Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr 20 25 30 -169- This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling in this Page)-Loading ·

、1T 1221482 A7B7 五、發明説明（167) 經濟部中央標準局員工消費合作社印製 GAC CCA GAA ACC GGA CGT CAG CTC TTG TGT GAC AAA TGT GCT CCT GGC Asp Pro Glu Thr Gly Arg Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly 35 40 45 ACC TAG CTA AAA CAG CAC TGC ACA GTC AGG AGG AAG ACA CTG TGT GTC Thr Tyr Leu Lys Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val 50 55 60 CCT TGC CCT GAC TAC TCT TAT ACA GAC AGC TGG CAC ACG AGT GAT GAA Pro Cys Pro Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu 65 70 75 TGC GTG TAC TGC AGC CCC GTG TGC AAG GAA CTG CAG ACC GTG AAA CAG Cys Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Thr Val Lys Gin 80 85 90 95 GAG TGC AAC CGC ACC CAC AAC CGA GTG TGC GAA TGT GAG GAA GGG CGC Glu Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Glu Glu Gly Arg 100 105 110 TAC CTG GAG CTC GAA TTC TGC TTG AAG CAC CGG AGC TGT CCC CCA GGC Tyr Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly 115 120 125 TTG GGT GTG CTG CAG GCT GGG ACC CCA GAG CGA AAC ACG GTT TGC AAA Leu Gly Val Leu Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys 130 ' 135 140 AGA TGT CCG GAT GGG TTC TTC TCA GGT GAG ACG TCA TCG AAA GCA CCC Arg Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro 145 150 155 TGT AGG AAA CAC ACC AAC TGC AGC TCA CTT GGC CTC CTG CTA ATT CAG Cys Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu lie Gin 160 165 170 175 AAA GGA AAT GCA ACA CAT GAC AAT GTA TGT TCC GGA AAC AGA GAA GGA Lys Gly Asn Ala Thr His Asp Asn Val Cys Ser Gly Asn Arg Glu Ala 180 185 190 ACT CAA AAT TGT GGA ATA GAT GTC ACC CTG TGC GAA GAG GCA TTC TTC Thr Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe 195 200 205 AGG TTT GCT GTG CCT ACC AAG ATT ATA CCG AAT TGG CTG AGT GTT CTG Arg Phe Ala Val Pro Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu 210 215 220 GTG GAC AGT TTG CCT GGG ACC AAA GTG AAT GCA GAG AGT GTA GAG AGG Val Asp Ser Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg 225 230 235170- 4 6 2 2 1 3 ο 6 3 8 ο 4 6 5 4 ο 5 2 5 5 ο ο 6 8 4 6 6 9 6 4 4 2 9 ο 4 8 (請先聞讀背面之注意事項再填寫本頁) -擊· 、11 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7B7 經濟部中央標準局員工消費合作社印製五、發明説明（168) ATA AAA CGG AGA CAC AGC TCG CAA GAG CAA ACT TTC CAG CTA CTT AAG 888 lie Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys 240 245 250 255 CTG TGG AAG CAT CAA AAC AGA GAC CAG GAA ATG GTG AAG AAG ATC ATC 936、 1T 1221482 A7B7 V. Description of invention (167) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, printed by GAC CCA GAA ACC GGA CGT CAG CTC TTG TGT GAC AAA TGT GCT CCT GGC Asp Pro Glu Thr Gly Arg Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly 35 40 45 ACC TAG CTA AAA CAG CAC TGC ACA GTC AGG AGG AAG ACA CTG TGT GTC Thr Tyr Leu Lys Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val 50 55 60 CCT TGC CCT GAC TAC TCT TAT ACA GAC AGC TGG CAC ACG AGT GAT GAA Pro Cys Pro Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu 65 70 75 TGC GTG TAC TGC AGC CCC GTG TGC AAG GAA CTG CAG ACC GTG AAA CAG Cys Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Thr Val Lys Gin 80 85 90 95 GAG TGC AAC CGC ACC CAC AAC CGA GTG TGC GAA TGT GAG GAA GGG CGC Glu Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Glu Glu Gly Arg 100 105 110 TAC CTG GAG CTC GAA TTC TGC TTG AAG CAC CGG AGC TGT CCC CCA GGC Tyr Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly 115 120 125 TTG GGT GTG CTG CAG GCT GGG ACC CCA GAG CGA AAC ACG GTT TGC AAA Leu GlyVal Leu Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys 130 '135 140 AGA TGT CCG GAT GGG TTC TTC TCA GGT GAG ACG TCA TCG AAA GCA CCC Arg Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Lys Ala Pro 145 150 155 TGT AGG AAA CAC ACC AAC TGC AGC TCA CTT GGC CTC CTG CTA ATT CAG Cys Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu lie Gin 160 165 170 175 AAA GGA AAT GCA ACA CAT GAC AAT GTA TGT TCC GGA AAC AGA GAA GGA Lys Gly Asn Ala Thr His Asp Asn Val Cys Ser Gly Asn Arg Glu Ala 180 185 190 ACT CAA AAT TGT GGA ATA GAT GTC ACC CTG TGC GAA GAG GCA TTC TTC Thr Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe 195 200 205 AGG TTT GCT GTG CCT ACC AAG ATT ATA CCG AAT TGG CTG AGT GTT CTG Arg Phe Ala Val Pro Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu 210 215 220 GTG GAC AGT TTG CCT GGG ACC AAA GTG AAT GCA GAG AGT GTA GAG AGG Val Asp Ser Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg 225 230 235170- 4 6 2 2 1 1 ο 6 3 8 ο 4 6 5 4 ο 5 2 5 5 ο ο 6 8 4 6 6 9 6 4 4 2 9 ο 4 8 (Please First read the notes on the back and then fill out this page)-Click · 11 This paper size applies Chinese National Standard (CNS) A4 (210X 297 mm) 1221482 A7B7 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Explanation (168) ATA AAA CGG AGA CAC AGC TCG CAA GAG CAA ACT TTC CAG CTA CTT AAG 888 lie Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys 240 245 250 255 CTG TGG AAG CAT CAA AAC AGA GAC CAG GAA ATG GTG AAG AAG ATC ATC 936

Leu Trp Lys His Gin Asn Arg Asp Gin Glu Met Val Lys Lys lie lie 260 265 270 CAA GAC ATT GAC CTC TGT GAA AGC AGT GTG CAA CGG CAT ATC GGC CAC 984Leu Trp Lys His Gin Asn Arg Asp Gin Glu Met Val Lys Lys lie lie 260 265 270 CAA GAC ATT GAC CTC TGT GAA AGC AGT GTG CAA CGG CAT ATC GGC CAC 984

Gin Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His lie Gly His 275 280 285 GCG AAC GTC ACC ACA GAG CAG CTC CGC ATC TTG ATG GAG AGC TTG CCT 1032Gin Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His lie Gly His 275 280 285 GCG AAC GTC ACC ACA GAG CAG CTC CGC ATC TTG ATG GAG AGC TTG CCT 1032

Ala Asn Leu Thr Thr Glu Gin Leu Arg lie Leu Met Glu Ser Leu Pro 290 295 300 φ GGG AAG AAG ATC AGC CCA GAC GAG ATT GAG AGA ACG AGA AAG ACC TGC 1080Ala Asn Leu Thr Thr Glu Gin Leu Arg lie Leu Met Glu Ser Leu Pro 290 295 300 φ GGG AAG AAG ATC AGC CCA GAC GAG ATT GAG AGA ACG AGA AAG ACC TGC 1080

Gly Lys Lys lie Ser Pro Asp Glu lie Glu Arg Thr Arg Lys Thr Cys 305 310 315 AAA CCC AGC GAG CAG CTC CTG AAG CTA CTG AGC TTG TGG AGG ATC AAA 1128Gly Lys Lys lie Ser Pro Asp Glu lie Glu Arg Thr Arg Lys Thr Cys 305 310 315 AAA CCC AGC GAG CAG CTC CTG AAG CTA CTG AGC TTG TGG AGG ATC AAA 1128

Lys Pro Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys 320 325 330 335 AAT GGA GAC CAA GAC ACC TTG AAG GGC CTG ATG TAC GCA CTC AAG CAC 1176Lys Pro Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys 320 325 330 335 AAT GGA GAC CAA GAC ACC TTG AAG GGC CTG ATG TAC GCA CTC AAG CAC 1176

Asn Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His 340 345 350 TTG AAA GCA TAC GAC TTT CCC AAA ACC GTC ACC CAC AGT CTG AGG AAG 1224Asn Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His 340 345 350 TTG AAA GCA TAC GAC TTT CCC AAA ACC GTC ACC CAC AGT CTG AGG AAG 1224

Leu Lys Ala Tyr His Phe Pro Lys Thr Val Thr His Ser Leu Arg Lys 355 360 365 ACC ATC AGG TTC TTG CAC AGC TTC ACC ATG TAC CGA TTG TAT CAG AAA 1272Leu Lys Ala Tyr His Phe Pro Lys Thr Val Thr His Ser Leu Arg Lys 355 360 365 ACC ATC AGG TTC TTG CAC AGC TTC ACC ATG TAC CGA TTG TAT CAG AAA 1272

Thr lie Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys 370 375 380 CTC TTT CTA GAA ATG ATA GGG AAT CAG GTT CAA TCA GTG AAG ATA AGC 1320Thr lie Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys 370 375 380 CTC TTT CTA GAA ATG ATA GGG AAT CAG GTT CAA TCA GTG AAG ATA AGC 1320

Leu Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser 385 390 395 TGC TTA TAGTTAGGAA TGGTCACTGG GCTGTTTCTT CAGGATGGGC CAACACTGAT 1376Leu Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser 385 390 395 TGC TTA TAGTTAGGAA TGGTCACTGG GCTGTTTCTT CAGGATGGGC CAACACTGAT 1376

Cys Leu 400 GGAGCAGATG GCTGCTTCTC CGGCTCTTGA AATGGCAGTT GATTCCTTTC TCATCAGTTG 1436 GTGGGAATGA AGATCCTCCA GCCCAACACA CACACTGGGG AGTCTGAGTC AGGAGAGTGA 1496 GGCAGGCTAT TTGATAATTG TGCAAAGCTG CCAGGTGTAC ACCTAGAAAG TCAAGCACCC 1556 -171 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝· 訂Cys Leu 400 GGAGCAGATG GCTGCTTCTC CGGCTCTTGA AATGGCAGTT GATTCCTTTC TCATCAGTTG 1436 GTGGGAATGA AGATCCTCCA GCCCAACACA CACACTGGGG AGTCTGAGTC AGGAGAGTGA 1496 GGCAGGCTAT TGCATAATTG TGCAAAGCTG CCAGGTGCAC ACCTAGC China ACCTAGC (Please fill in this page for attention)

2 8 4 2 2 I A7B7 五、發明説明（169)2 8 4 2 2 I A7B7 V. Description of the invention (169)

TGAGAAAGAGTGAGAAAGAG

TGAGTACTCATGAGTACTCA

TTATTTTTTTTTATTTTTTT

GCAAGTGCTCGCAAGTGCTC

TGATAGTCTATGATAGTCTA

TTGTAGGTTTTTGTAGGTTT

TTGCAGACTTTTGCAGACTT

CAGGAGTCCACAGGAGTCCA

AGTATGAAAAAGTATGAAAA

GGGCACTAAAGGGCACTAAA

CCCAATAGTTCCCAATAGTT

TTACTGCATGTTACTGCATG

CTCCATTGGACTCCATTGGA

GTGTTAAAGGGTGTTAAAGG

AAGACTATTAAAGACTATTA

GATATTTTTAGATATTTTTA

GAAGGCTTCTGAAGGCTTCT

ATTCTTTTTTATTCTTTTTT

TACCACTGAGTACCACTGAG

TGACATTCTTTGACATTCTT

CTAGGCAAGTCTAGGCAAGT

GGCTAGACAAGGCTAGACAA

GTGTTTCTTGGTGTTTCTTG

ATAATCAACAATAATCAACA

AGAAACTACTAGAAACTACT

TATCCAGCTGTATCCAGCTG

CAGTAATTCACAGTAATTCA

TCTCTCTGAATCTCTCTGAA

CTTTTATTAACTTTTATTAA

CAGTATTGCTCAGTATTGCT

TAACCTCAAATAACCTCAAA

ACTATCTTCTACTATCTTCT

TCGGAGCTGGTCGGAGCTGG

CTAAATCTCCCTAAATCTCC

TTTTCTACAATTTTCTACAA

TGACCGTTAGTGACCGTTAG

GCAGGGGTAGGCAGGGGTAG

TTCCTCTGTATTCCTCTGTA

AATTTTATTCAATTTTATTC

ATATGGAGAAATATGGAGAA

TCATGCCTGGTCATGCCTGG

ACTGGAAATAACTGGAAATA

TATGGGAATATATGGGAATA

AAAGCTGATGAAAGCTGATG

ATTTATATCCATTTATATCC

CATAGGCCCTCATAGGCCCT

GTGTCATCCCGTGTCATCCC

GGACCGAACCGGACCGAACC

AACCCCTGAAAACCCCTGAA

TTCGTATCAGTTCGTATCAG

CTATTTTTCCCTATTTTTCC

GTTATGGTAGGTTATGGTAG

GTTGTACCTAGTTGTACCTA

CTTCTATCAACTTCTATCAA

AGAATTGATAAGAATTGATA

TTCAGTGTCTTTCAGTGTCT

GTAATAATAAGTAATAATAA

TCTAACTTAATCTAACTTAA

CTCTTCTGTACTCTTCTGTA

ATCCAGATCCAG

TTCCTTCCTCTTCCTTCCTC

TAGATGAAGGTAGATGAAGG

CAGGGCCTTGCAGGGCCTTG

GGCCTCTTTCGGCCTCTTTC

GTGCACGAGCGTGCACGAGC

CTCTGAAGATCTCTGAAGAT

TTTATTTAACTTTATTTAAC

AGCTGACTCCAGCTGACTCC

CATTGGCTAGCATTGGCTAG

TTGCCCCCAATTGCCCCCAA

ACTGACTATGACTGACTATG

TAATAGAAATTAATAGAAAT

GAAGCTTTGAGAAGCTTTGA

AAAGTTACTAAAAGTTACTA

TCCTTATGGATCCTTATGGA

CCTCTTTTATCCTCTTTTAT

CGCTTGCGAGCGCTTGCGAG

TTTCTGCCTCTTTCTGCCTC

CTTATCCCATCTTATCCCAT

TTGATTCGAGTTGATTCGAG

AGACTGCCACAGACTGCCAC

AAGTACATTTAAGTACATTT

CTTTGTTTCACTTTGTTTCA

CGTTCAACAACGTTCAACAA

CGCCCTCTTACGCCCTCTTA

AAAATCTAGAAAAATCTAGA

GATTTCAGTTGATTTCAGTT

ATATATCTGT 1616 1676 1736 1796 1856 1916 1976 2036 2096 2156 2216 2276 2336 2396 2432 (請先閲讀背面之注意事項再填寫本頁) -裝· 訂經濟部中央標準局員工消費合作社印製 (2) SEQ ID NO ·· 121的資訊： (i)序列特性： (A) 長度：401胺基酸 (B) 類別··胺基酸 (D)拓樸學：線型 (H)分子類別··蛋白質 (xi)序列説明：SEQ ID NO ·· 121 :ATATATCTGT 1616 1676 1736 1796 1856 1916 1976 2036 2096 2156 2216 2276 2336 2396 2432 (Please read the precautions on the back before filling out this page)-Binding · Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (2) SEQ ID NO · · 121 information: (i) sequence characteristics: (A) length: 401 amino acids (B) category · amino acids (D) topology: linear (H) molecular category · protein (xi) sequence description: SEQ ID NO: 121:

Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Phe Leu Asp He He 15 10 15Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Phe Leu Asp He He 15 10 15

Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 •172 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（170)Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 • 172 This paper size applies to China National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs System five, description of the invention

Pro Glu Thr Gly Arg Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly Thr 35 40 45Pro Glu Thr Gly Arg Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly Thr 35 40 45

Tyr Leu Lys Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val Pro 50 55 60Tyr Leu Lys Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val Pro 50 55 60

Cys Pro Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80Cys Pro Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80

Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Thr Val Lys Gin Glu 85 90 95Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Thr Val Lys Gin Glu 85 90 95

Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Glu Glu Gly Arg Tyr 100 105 110Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Glu Glu Gly Arg Tyr 100 105 110

Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Leu 115 120 125Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Leu 115 120 125

Gly Val Leu Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140Gly Val Leu Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140

Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160

Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu. lie Gin Lys 165 170 175Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu. Lie Gin Lys 165 170 175

Gly Asn Ala Thr His Asp Asn Val Cys Ser Gly Asn Arg Glu Ala Thr 180 185 190Gly Asn Ala Thr His Asp Asn Val Cys Ser Gly Asn Arg Glu Ala Thr 180 185 190

Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205

Phe Ala Val Pro Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu Val 210 215 220Phe Ala Val Pro Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu Val 210 215 220

Asp Ser Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240Asp Ser Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240

Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255

Trp Lys His Gin Asn Arg Asp Gin Glu Met Val Lys Lys lie lie Gin 260 265 270Trp Lys His Gin Asn Arg Asp Gin Glu Met Val Lys Lys lie lie Gin 260 265 270

Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His lie Gly His Ala 275 280 285Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His lie Gly His Ala 275 280 285

Asn Leu Thr Thr Glu Gin Leu Arg lie Leu Met Glu Ser Leu Pro Gly 290 295 300 -173- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） (請先閲讀背面之注意事項再填寫本頁) -裝. 、?τ 1221482 A7B7 經濟部中央標準局員工消費合作社印製五、發明説明（171)Asn Leu Thr Thr Glu Gin Leu Arg lie Leu Met Glu Ser Leu Pro Gly 290 295 300 -173- This paper size applies to China National Standard (CNS) Α4 size (210 × 297 mm) (Please read the precautions on the back before filling in this (Page)-Equipment.,? Τ 1221482 A7B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the Invention (171)

Lys Lys lie Ser Pro Asp Glu lie Glu Arg Thr Arg Lys Thr Cys Lys 305 310 315 320Lys Lys lie Ser Pro Asp Glu lie Glu Arg Thr Arg Lys Thr Cys Lys 305 310 315 320

Pro Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335 - Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu 340 345 350Pro Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335-Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu 340 345 350

Lys Ala Tyr His Phe Pro Lys Thr Val Thr His Ser Leu Arg Lys Thr 355 360 365Lys Ala Tyr His Phe Pro Lys Thr Val Thr His Ser Leu Arg Lys Thr 355 360 365

He Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu 370 375 380He Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu 370 375 380

Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys 385 390 395 400Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys 385 390 395 400

Leu (2) SEQ ID NO ·· 122的資訊·· (i) 序列特性： (A)長度：1 324鹼對 • (B)類別：核酸 (C) 股型··單股 (D) 拓樸學：線型 (ii) 分子類別：cDNA (ix)特徵：Leu (2) SEQ ID NO · 122 information · (i) Sequence characteristics: (A) Length: 1 324 base pairs · (B) Category: Nucleic acid (C) Strand type · Single strand (D) Topology : Linear (ii) Molecular Class: cDNA (ix) Features:

(A) 名稱/關鍵詞：CDS (B) 位置：90···1292 (xi)序列説明：SEQ ID NO : 122 : CCTTATATAA ACGTCATGAT TGCCTGGGCT GCAGAGACGC ACCTAGCACT GACCCAGCGG 60 CTGCCTCCTG AGGTTTCCCG AGGACCACA ATG AAC AAG TGG CTG TGC TGC GCA 113(A) Name / Keyword: CDS (B) Location: 90 ... 1292 (xi) Sequence description: SEQ ID NO: 122: CCTTATATAA ACGTCATGAT TGCCTGGGCT GCAGAGACGC ACCTAGCACT GACCCAGCGG 60 CTGCCTCCTG AGGTTTCCCG AGGACCACA ATG AAC AAG TGG CTG TGC TGC GCA 113

Met Asn Lys Trp Leu Cys Cys Ala 1 5 CTC CTG GTG CTC CTG GAC ATC ATT GAA TGG ACA ACC CAG GAA ACC CTT 161Met Asn Lys Trp Leu Cys Cys Ala 1 5 CTC CTG GTG CTC CTG GAC ATC ATT GAA TGG ACA ACC CAG GAA ACC CTT 161

Leu Leu Val Leu Leu Asp lie lie Glu Trp Thr Thr Gin Glu Thr Leu 10 15 20 -174- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公襲） (請先閲讀背面之注意事項再填寫本頁) •裝· 訂 1221482 A7 B7 五、發明説明（172) 經濟部中央標準局員工消費合作社印製 CCT CCA AAG TAC TTG CAT TAT GAC CCA GAA ACT GGT CAT CAG CTC CTG 209 Pro Pro Lys Tyr Leu His Tyr Asp Pro Glu Thr Gly His Gin Leu Leu 25 30 35 40 TGT GAC AAA TGT GCT CCT GGC ACC TAC CTA AAA CAG CAC TGC ACA GTG 257 Cys Asp Lys Cys Ala Pro Gly Thr Tyr Leu Lys Gin His Cys Thr Val 45 50 55 AGG AGG AAG ACA TTG TGT GTC CCT TGC CCT GAC CAC TCT TAT ACG GAC 305 Arg Arg Lys Thr Leu Cys Val Pro Cys Pro Asp His Ser Tyr Thr Asp 60 65 70 AGC TGG CAC ACC AGT GAT GAG TGT GTG TAT TGC AGC CCA GTG TGC AAG 353 Ser Trp His Thr Ser Asp Glu Cys Val Tyr Cys Ser Pro Val Cys Lys 75 80 85 GAA CTG CAG TCC GTG AAG CAG GAG TGC AAC CGC ACC CAC AAC CGA GTG 401 Glu Leu Gin Ser Val Lys Gin Glu Cys Asn Arg Thr His Asn Arg Val 90 95 100 TGT GAG TGT GAG GAA GGG CGT TAC CTG GAG ATC GAA TTC TGC TTG AAG 449 Cys Glu Cys Glu Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu Lys 105 110 115 120 CAC CGG AGC TGT cqc CCG GGC TCC GGC GTG GTG CAA GCT GGA ACC CCA 497 Hl3 Arg Ser Cys Pro Pro Gly Ser Gly Val Val Gin Ala Gly Thr Pro 125 130 135 GAG CGA AAC ACA GTT TGC AAA AAA TGT CCA GAT GGG TTC TTC TCA GGT 545 Glu Arg Asn Thr Val Cys Lys Lys Cys Pro Asp Gly Phe Phe Ser Gly 140 145 150 GAG ACT TCA TCG AAA GCA CCC TGT ATA AAA CAC ACG AAC TGC AGC ACA 593 Glu Thr Ser Ser Lys Ala Pro Cys lie Lys His Thr Asn Cys Ser Thr 155 160 165 TTT GGC CTC CTG CTA ATT CAG AAA Lys GGA AAT GCA ACA CAT GAC AAC GTG 641 Phe Gly Leu Leu Leu lie Gin Gly Asn Ala Thr His Asp Asn Val 170 175 180 TGT TCC GGA AAC AGA GAA GCC ACG CAA AAG TGT GGA ATA GAT GTC ACC 689 Cys Ser Gly Asn Arg Glu Ala Thr Gin Lys Cys Gly lie Asp Val Thr 185 190 195 200 CTG TGT GAA GAG GCC TTC TTC AGG TTT GCT GTT CCT ACC AAG ATT ATA 737 Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys lie lie 205 210 215 (請先閲讀背面之注意事項再填寫本頁) 裝·Leu Leu Val Leu Leu Asp lie lie Glu Trp Thr Thr Gin Glu Thr Leu 10 15 20 -174- This paper size applies to China National Standard (CNS) A4 specification (210X297 public attack) (Please read the precautions on the back before filling in this Page) • Binding · Binding 1221482 A7 B7 V. Description of Invention (172) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs CCT CCA AAG TAC TTG CAT TAT GAC CCA GAA ACT GGT CAT CAG CTC CTG 209 Pro Pro Lys Tyr Leu His Tyr Asp Pro Glu Thr Gly His Gin Leu Leu 25 30 35 40 TGT GAC AAA TGT GCT CCT GGC ACC TAC CTA AAA CAG CAC TGC ACA GTG 257 Cys Asp Lys Cys Ala Pro Gly Thr Tyr Leu Lys Gin His Cys Thr Val 45 50 55 AGG AGG AAG ACA TTG TGT GTC CCT TGC CCT GAC CAC TCT TAT ACG GAC 305 Arg Arg Lys Thr Leu Cys Val Pro Cys Pro Asp His Ser Tyr Thr Asp 60 65 70 AGC TGG CAC ACC AGT GAT GAG TGT GTG TAT TGC AGC CCA GTG TGC AAG 353 Ser Trp His Thr Ser Asp Glu Cys Val Tyr Cys Ser Pro Val Cys Lys 75 80 85 GAA CTG CAG TCC GTG AAG CAG GAG TGC AAC CGC ACC CAC AAC CGA GTG 401 Glu Leu Gin Ser Val Lys Gin Glu Cys Asn Arg Thr His Asn Arg Val 90 95 100 TGT GAG TGT GAG GAA GGG CGT TAC CTG GAG ATC GAA TTC TGC TTG AAG 449 Cys Glu Cys Glu Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu Lys 105 110 115 120 CAC CGG AGC TGT cqc CCG GGC TCC GGC GTG GTG CAA GCT GGA ACC CCA 497 Hl3 Arg Ser Cys Pro Pro Gly Ser Gly Val Val Gin Ala Gly Thr Pro 125 130 135 GAG CGA AAC ACA GTT TGC AAA AAA TGT CCA GAT GGG TTC TTC TCA GGT 545 Glu Arg Asn Thr Val Cys Lys Lys Cys Pro Asp Gly Phe Phe Ser Gly 140 145 150 GAG ACT TCA TCG AAA GCA CCC TGT ATA AAA CAC ACG AAC TGC AGC ACA 593 Glu Thr Ser Ser Lys Ala Pro Cys lie Lys His Thr Asn Cys Ser Thr 155 160 165 TTT GGC CTC CTG CTA ATT CAG AAA Lys GGA AAT GCA ACA CAT GAC AAC GTG 641 Phe Gly Leu Leu Leu lie Gin Gly Asn Ala Thr His Asp Asn Val 170 175 180 TGT TCC GGA AAC AGA GAA GCC ACG CAA AAG TGT GGA ATA GAT GTC ACC 689 Cys Ser G ly Asn Arg Glu Ala Thr Gin Lys Cys Gly lie Asp Val Thr 185 190 195 200 CTG TGT GAA GAG GCC TTC TTC AGG TTT GCT GTT CCT ACC AAG ATT ATA 737 Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys lie lie 205 210 215 (Please read the notes on the back before filling this page)

、1T -175- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐) 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（173) CCA AAT TGG CTG AGT GTT TTG GTG GAC AGT TTG CCT GGG ACC AAA GTG 785、 1T -175- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (173) CCA AAT TGG CTG AGT GTT TTG GTG GAC AGT TTG CCT GGG ACC AAA GTG 785

Pro Asn Trp Leu Ser Val Leu Val Asp Ser Leu Pro Gly Thr Lys Val 220 225 230 AAT GCC GAG AGT GTA GAG AGG ATA AAA CGG AGA CAC AGC TCA CAA GAG 833Pro Asn Trp Leu Ser Val Leu Val Asp Ser Leu Pro Gly Thr Lys Val 220 225 230 AAT GCC GAG AGT GTA GAG AGG ATA AAA CGG AGA CAC AGC TCA CAA GAG 833

Asn Ala Glu Ser Val Glu Arg lie Lys Arg Arg His Ser Ser Gin Glu 235 240 245 CAA ACC TTC CAG CTG CTG AAG CTG TGG AAA CAT CAA AAC AGA GAC CAG 881Asn Ala Glu Ser Val Glu Arg lie Lys Arg Arg His Ser Ser Gin Glu 235 240 245 CAA ACC TTC CAG CTG CTG AAG CTG TGG AAA CAT CAA AAC AGA GAC CAG 881

Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn Arg Asp Gin 250 255 260 GAA ATG GTG AAG AAG ATC ATC CAA GAC ATT GAC CTC TGT GAA AGC AGC 929Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn Arg Asp Gin 250 255 260 GAA ATG GTG AAG AAG ATC ATC CAA GAC ATT GAC CTC TGT GAA AGC AGC 929

Glu Met Val Lys Lys lie lie Gin Asp lie Asp Leu Cys Glu Ser Ser 265 270 275 280 GTG CAG CGG CAT CTC GGC CAC TCG AAC GTC ACC ACA GAG CAG CTT CTT · 977Glu Met Val Lys Lys lie lie Gin Asp lie Asp Leu Cys Glu Ser Ser 265 270 275 280 GTG CAG CGG CAT CTC GGC CAC TCG AAC GTC ACC ACA GAG CAG CTT CTT · 977

Val Gin Arg His Leu Gly His Ser Asn Leu Thr Thr Glu Gin Leu Leu 285 290 295 GCC TTG ATG GAG AGC CTG CCT GGG AAG AAG ATC AGC CCA GAA GAG ATT 1025Val Gin Arg His Leu Gly His Ser Asn Leu Thr Thr Glu Gin Leu Leu 285 290 295 GCC TTG ATG GAG AGC CTG CCT GGG AAG AAG ATC AGC CCA GAA GAG ATT 1025

Ala Leu Met Glu Ser Leu Pro Gly Lys Lys lie Ser Pro Glu Glu lie 300 305 310 GAG AGA ACG AGA AAG ACC TGC AAA TCG AGC GAG CAG CTC CTG AAG CTA 1073Ala Leu Met Glu Ser Leu Pro Gly Lys Lys lie Ser Pro Glu Glu lie 300 305 310 GAG AGA ACG AGA AGA ACC TGC AAA TCG AGC GAG CAG CTC CTG AAG CTA 1073

Glu Arg Thr Arg Lys Thr Cys Lys Ser Ser Glu Gin Leu Leu Lys Leu 315 320 325 CTC AGT TTA TGG AGG ATC AAA AAT GGT GAC CAA GAC ACC TTG AAG GGC 1121Glu Arg Thr Arg Lys Thr Cys Lys Ser Ser Glu Gin Leu Leu Lys Leu 315 320 325 CTC AGT TTA TGG AGG ATC AAA AAT GGT GAC CAA GAC ACC TTG AAG GGC 1121

Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys Gly 330 335 340 CTG ATG TAT GCC CTC AAG CAC TTG AAA ACA TCC CAC TTT CCC AAA ACT 1169Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys Gly 330 335 340 CTG ATG TAT GCC CTC AAG CAC TTG AAA ACA TCC CAC TTT CCC AAA ACT 1169

Leu Met Tyr Ala Leu Lys His Leu Lys Thr Ser His Phe Pro Lys Thr 345 350 355 360 GTC ACC CAC AGT CTG AGG AAG ACC ATG AGG TTC CTG CAC AGC TTC ACA 1217Leu Met Tyr Ala Leu Lys His Leu Lys Thr Ser His Phe Pro Lys Thr 345 350 355 360 GTC ACC CAC AGT CTG AGG AAG ACC ATG AGG TTC CTG CAC AGC TTC ACA 1217

Val Thr His Ser Leu Arg Lys Thr Met Arg Phe Leu His Ser Phe Thr 365 370 375 ATG TAC AGA CTG TAT CAG AAG CTC TTT TTA GAA ATG ATA GGG AAT CAG 1265Val Thr His Ser Leu Arg Lys Thr Met Arg Phe Leu His Ser Phe Thr 365 370 375 ATG TAC AGA CTG TAT CAG AAG CTC TTT TTA GAA ATG ATA GGG AAT CAG 1265

Met Tyr Arg Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn Gin 380 385 390 GTT CAA TCC GTG AAA ATA AGC TGC TTA TAACTAGGAA TGGTCACTGG 1312Met Tyr Arg Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn Gin 380 385 390 GTT CAA TCC GTG AAA ATA AGC TGC TTA TAACTAGGAA TGGTCACTGG 1312

Val Gin Ser Val Lys lie Ser Cys Leu 395 400 GCTGTTTCTT CA 1324 -176· 本紙張尺度適用中國國家標準（CNS ) Α4規格（2!〇Χ297公羡） (請先閲讀背面之注意事項再填寫本頁) •裝. 、?τ 1221482 A7B7 經濟部中央標準局員工消費合作社印製五、發明説明（174) (2) SEQ ID NO : 123的資訊： (i) 序列特性： (A) 長$ : 401胺基酸 (B) 類‘：胺基酸 (D)拓樸學：線型 (ii) 分子類別：蛋白質 (xi)序列説明：SEQ ID NO : 123 :Val Gin Ser Val Lys lie Ser Cys Leu 395 400 GCTGTTTCTT CA 1324 -176 · This paper size applies to China National Standard (CNS) Α4 specification (2! 〇 × 297 public envy) (Please read the precautions on the back before filling this page) • Equipment.,? Τ 1221482 A7B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the Invention (174) (2) Information of SEQ ID NO: 123: (i) Sequence characteristics: (A) Long $: 401 amine Basic acid (B) class': Amino acid (D) Topology: Linear (ii) Molecular class: Protein (xi) Sequence description: SEQ ID NO: 123:

Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Leu Leu Asp lie lie 1 5 10 15Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Leu Leu Asp lie lie 1 5 10 15

Glu Trp Thr Thr Gin Glu Thr Leu Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30Glu Trp Thr Thr Gin Glu Thr Leu Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30

Pro Glu Thr Gly His Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly Thr 35 40 45Pro Glu Thr Gly His Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly Thr 35 40 45

Cys Pro Asp His Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80Cys Pro Asp His Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80

Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Ser Val Lys Gin Glu 85 90 95Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Ser Val Lys Gin Glu 85 90 95

Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Ser 115 120 125Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Ser 115 120 125

Gly Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Lys 130 135 140Gly Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Lys 130 135 140

Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 lie Lys His Thr Asn Cys Ser Thr Phe Gly Leu Leu Leu lie Gin Lys 165 170 175Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 lie Lys His Thr Asn Cys Ser Thr Phe Gly Leu Leu Leu lie Gin Lys 165 170 175

Gly Asn Ala Thr His Asp Asn Val Gys Ser Gly Asn Arg Glu Ala Thr 180 185 190Gly Asn Ala Thr His Asp Asn Val Gys Ser Gly Asn Arg Glu Ala Thr 180 185 190

Gin Lys Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 177- 本紙張尺度適用中國國家標準（CNS ) Α4規格（21〇Χ297公釐） ---------'^裝-- (請先閲讀背面之注意事項再填寫本頁)Gin Lys Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 177- This paper size applies the Chinese National Standard (CNS) A4 specification (21〇 × 297 mm) --------- ' ^ Install-(Please read the notes on the back before filling this page)

、1T 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（175)1T 1221482 A7 B7 Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs V. Description of Invention (175)

Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His Leu Gly His Ser 275 280 285Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His Leu Gly His Ser 275 280 285

Asn Leu Thr Thr Glu Gin Leu Leu Ala Leu Met Glu Ser Leu Pro Gly 290 295 300Asn Leu Thr Thr Glu Gin Leu Leu Ala Leu Met Glu Ser Leu Pro Gly 290 295 300

Lys Lys lie Ser Pro Glu Glu lie Glu Arg Thr Arg Lys Thr Cys Lys 305 310 315 320Lys Lys lie Ser Pro Glu Glu lie Glu Arg Thr Arg Lys Thr Cys Lys 305 310 315 320

Ser Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335Ser Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335

Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu 340 345 350Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu 340 345 350

Lys Thr Ser His Phe Pro Lys Thr Val Thr His Ser Leu Arg Lys Thr 355 360 365Lys Thr Ser His Phe Pro Lys Thr Val Thr His Ser Leu Arg Lys Thr 355 360 365

Met Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu 370 375 380Met Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu 370 375 380

Leu (2) SEQ ID NO : 124的資訊： (i) 序列特性： (A) 長度：1355鹼對 (B) 類別··核酸 (C) 股型：單股 (D) 拓樸學：線型Leu (2) Information of SEQ ID NO: 124: (i) Sequence characteristics: (A) Length: 1355 base pairs (B) Category · Nucleic acid (C) Strand type: Single strand (D) Topology: Linear type

(ii) 分子類別：cDNA -178· 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) -裝· 1221482 A7B7 經濟部中央標準局員工消費合作社印製五、發明説明（176) (ix)特徵： (A) 名稱/關鍵詞：CDS (B) 位置：94···1296 (xi)序列説明：SEQ ID NO ·· 124 ·· GTATATATAA CGTGATGAGC GTACGGGTGC GGAGACGCAC CGGAGCGCTC GCCCAGCCGC CGCTCCAAGC CCCTGAGGTT TCCGGGGACC ACA ATG AAC AAG TTG CTG TGC TGC Met Asn Lys Leu Leu Cys Cys 1 5 GCG CTC GTG TTT CTG GAC ATC TCC ATT AAG TGG ACC ACC CAG GAA ACG Ala Leu Val Phe Leu Asp lie Ser lie Lys Trp Thr Thr Gin Glu Thr 10 15 20 TTT CCT CCA AAG TAC CTT CAT TAT GAC GAA GAA ACC TCT CAT CAG CTG Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gin Leu 25 30 35 TTG TGT GAC AAA TGT CCT CCT GGT ACC TAC CTA AAA CAA CAC TGT ACA Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gin His Cys Thr 40 45 50 55 GCA AAG TGG AAG ACC GTG TGC GCC CCT TGC CCT GAC CAC TAC TAC ACA Ala Lys Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr 60 65 70 GAC AGC TGG CAC ACC AGT GAC GAG TGT CTA TAC TGC AGC CCC GTG TGC Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys 75 80 85 AAG GAG CTG CAG TAC GTC AAG CAG GAG TGC AAT CGC ACC CAC AAC CGC Lys Glu Leu Gin Tyr Val Lys Gin Glu Cys Asn Arg Thr His Asn Arg 90 95 100 GTG TGC GAA TGC AAG GAA GGG CGC TAC CTT GAG ATA GAG TTC TGC TTG Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu 105 110 115 AAA CAT AGG AGC TGC CCT CCT GGA TTT GGA GTG GTG CAA GCT GGA ACC Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gin Ala Gly Thr 120 125 130 135 CCA GAG CGA AAT ACA GTT TGC AAA AGA TGT CCA GAT GGG TTC TTC TCA Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser 140 145 150 ο 6 (請先閲讀背面之注意事項再填寫本頁) -裝·(ii) Molecular type: cDNA -178 · This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page)-Pack · 1221482 A7B7 Central Bureau of Standards, Ministry of Economic Affairs Printed by the Employee Consumption Cooperative. 5. Description of the invention (176) (ix) Features: (A) Name / Keyword: CDS (B) Location: 94 ··· 1296 (xi) Sequence description: SEQ ID NO ·· 124 ·· GTATATATAA CGTGATGAGC GTACGGGTGC GGAGACGCAC CGGAGCGCTC GCCCAGCCGC CGCTCCAAGC CCCTGAGGTT TCCGGGGACC ACA ATG AAC AAG TTG CTG TGC TGC TGC Met Asn Lys Leu Leu Leu Cys Cyp AUC TAC ATC TCC lie Lys Trp Thr Thr Gin Glu Thr 10 15 20 TTT CCT CCA AAG TAC CTT CAT TAT GAC GAA GAA GAA ACC TCT CAT CAG CTG Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gin Leu 25 30 35 TTG TGT GAC AAA TGT CCT CCT GGT ACC TAC CTA AAA CAA CAC TGT ACA Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gin His Cys Thr 40 45 50 55 GCA AAG TGG AAG ACC GTG TGC GCC CCT TGC CCT GAC CAC TAC TAC ACA Ala Lys Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr 60 65 70 GAC AGC TGG CAC ACC AGT GAC GAG TGT CTA TAC TGC AGC CCC GTG TGC Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys 75 80 85 AAG GAG CTG CAG TAC GTC AAG CAG GAG TGC AAT CGC ACC CAC AAC CGC Lys Glu Leu Gin Tyr Val Lys Gin Glu Cys Asn Arg Thr His Asn Arg 90 95 100 GTG TGC GAA TGC AAG GAA GGG CGC TAC CTT GAG ATA GAG TTC TGC TTG Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu 105 110 115 AAA CAT AGG AGC TGC CCT CCT GCT TGA GTT GTA GTG GTG CAA GCT GGA ACC Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gin Ala Gly Thr 120 125 130 135 CCA GAG CGA AAT ACA GTT TGC AAA AGA TGT CCA GAT GGG TTC TTC TCA Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser 140 145 150 ο 6 ( (Please read the notes on the back before filling out this page)

、1T 一 __-179-本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7B7 五、發明説明（177) AAT GAG ACG TCA TCT AAA GCA CCC TGT AGA AAA CAC ACA AAT TGC AGT 594 As η Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser 155 160 165 GTC TTT GGT CTC CTG CTA ACT CAG AAA Lys GGA AAT GCA ACA CAC GAC AAC 642 Val Phe Gly Leu Leu Leu Thr Gin Gly Asn Ala Thr His Asp Asn 170 175 180 經濟部中央標準局員工消費合作社印製 ATA TGT TCC GGA AAC AGT GAA TCA ACT CAA AAA TGT GGA ATA GAT GTT 690 lie Cys Ser Gly Asn Ser Glu Ser Thr Gin Lys Cys Gly lie Asp Val 185 190 195 ACC CTG TGT GAG GAG GCA TTC TTC AGG TTT GCT GTT CCT ACA AAG TTT 738 Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe 200 205 210 215 e ACG CCT AAC TGG CTT AGT GTC TTG GTA GAC AAT TTG CCT GGC ACC AAA 786 Thr Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys 220 225 230 GTA AAC GCA GAG AGT GTA GAG AGG ATA AAA CGG CAA CAC AGC TCA CAA 834 Val Asn Ala Glu Ser Val Glu Arg lie Lys Arg Gin His Ser Ser Gin 235 240 245 GAA CAG ACT TTC CAG CTG CTG AAG TTA TGG AAA CAT CAA AAC AAA GCC 882 Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn Lys Ala 250 255 260 CAA GAT ATA GTC AAG AAG ATC ATC CAA GAT ATT GAC CTC TGT GAA AAC 930 Gin Asp lie Val Lys Lys lie lie Gin Asp lie Asp Leu Cys Glu Asn 265 270 275 AGC GTG CAG CGG CAC ATT GGA CAT GCT AAC CTC ACC TTC GAG CAG CTT 978 Ser Val Gin Arg His lie Gly His Ala Asn Leu Thr Phe Glu Gin Leu 280 285 290 295 CGT AGC TTG ATG GAA AGC TTA CCG GGA AAG AAA GTG GGA GCA GAA GAC 1026 Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp 300 305 310 ATT GAA AAA ACA ATA AAG GCA TGC AAA CCC AGT GAC CAG ATC CTG AAG 1074 lie Glu Lys Thr lie Lys Ala Cys Lys Pro Ser Asp Gin lie Leu Lys 315 320 325 CTG CTC AGT TTG TGG CGA ATA AAA AAT GGC (SAC CAA GAC ACC TTG AAG 1122 Leu Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys 330 335 340 (請先閲讀背面之注意事項再填寫本頁) -裝· 訂 180· 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（178) GGC CTA ATG CAC GCA CTA AAG CAC TCA AAG ACG TAC CAC TTT CCC AAA 1170、 1T __- 179- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7B7 V. Description of invention (177) AAT GAG ACG TCA TCT AAA GCA CCC TGT AGA AAA CAC ACA AAT TGC AGT 594 As η Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser 155 160 165 GTC TTT GGT CTC CTG CTA ACT CAG AAA Lys GGA AAT GCA ACA CAC GAC AAC 642 Val Phe Gly Leu Leu Leu Thr Gin Gly Asn Ala Thr His Asp Asn 170 175 180 Printed by ATA TGT TCC GGA AAC AGT GAA TCA ACT CAA AAA TGT GGA ATA GAT GTT 690 lie Cys Ser Gly Asn Ser Glu Ser Thr Gin Lys Cys Gly lie Asp Val 185 190 195 ACC CTG TGT GAG GAG GCA TTC TTC AGG TTT GCT GTT CCT ACA AAG TTT 738 Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe 200 205 210 215 e ACG CCT AAC TGG CTT AGT GTC TTG GTA GAC AAT TTG CCT GGC ACC AAA 786 Thr Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys 220 225 230 GTA AAC GCA GAG AGT GTA GAG AGG ATA AAA CGG CAA CAC AGC TCA CAA 834 Val Asn Ala Glu Ser Val Glu Arg lie Lys Arg Gin His Ser Ser Gin 235 240 245 GAA CAG ACT TTC CAG CTG CTG AAG TTA TGG AAA CAT CAA AAC AAA GCC 882 Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn Lys Ala 250 255 260 CAA GAT ATA GTC AAG AAG ATC ATC CAA GAT ATT GAC CTC TGT GAA AAC 930 Gin Asp lie Val Lys Lys lie lie Gin Asp liep Leu Cys Glu Asn 265 270 275 AGC GTG CAG CGG CAC ATT GGA CAT GCT AAC CTC ACC TTC GAG CAG CTT 978 Ser Val Gin Arg His lie Gly His Ala Asn Leu Thr Phe Glu Gin Leu 280 285 290 295 CGT AGC TTG ATG GAA AGC TTA CCG GGA AAG AAA GTG GGA GCA GAA GAC 1026 Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp 300 305 310 ATT GAA AAA ACA ATA AAG GCA TGC AAA CCC AGT GAC CAG ATC CTG AAG 1074 lie Glu Lys Thr lie Lys Ala Cys Lys Pro Ser Asp Gin lie Leu Lys 315 320 325 CTG CTC AGT TTG TGG CGA ATA AAA AAT GGC (SAC CAA GAC ACC TTG AAG 1122 Leu Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys 330 335 340 (Please read the precautions on the back before filling out this page)-Binding 180 Book Paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1221482 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of invention (178) GGC CTA ATG CAC GCA CTA AAG CAC TCA AAG ACG TAC CAC TTT CCC AAA 1170

Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys 345 .350 355 ACT GTC ACT CAG AGT CTA AAG AAG ACC ATC AGG TTC CTT CAC AGC TTC 1218Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys 345 .350 355 ACT GTC ACT CAG AGT CTA AAG AAG ACC ATC AGG TTC CTT CAC AGC TTC 1218

Thr Val Thr Gin Ser Leu Lys Lys Thr lie Arg Phe Leu His Ser Phe 360 365 370 375 ACA ATG TAC AAA TTG TAT CAG AAG TTA TTT TTA GAA ATG ATA GGT AAC 1266Thr Val Thr Gin Ser Leu Lys Lys Thr lie Arg Phe Leu His Ser Phe 360 365 370 375 ACA ATG TAC AAA TTG TAT CAG AAG TTA TTT TTA GAA ATG ATA GGT AAC 1266

Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn 380 385 390 CAG GTC CAA TCA GTA AAA ATA AGC TGC TTA TAACTGGAAA TGGCCATTGA 1316Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn 380 385 390 CAG GTC CAA TCA GTA AAA ATA AGC TGC TTA TAACTGGAAA TGGCCATTGA 1316

Gin Val Gin Ser Val Lys lie Ser Cys Leu 395 400 GCTGTTTCCT CACAATTGGC GAGATCCCAT GGATGATAA 1355 (2) SEQ ID NO : 125的資訊： (i) 序列特性： (A) 長度：401胺基酸 (B) 類別：胺基酸 (D)拓樸學：線型 (ii) 分子類別：蛋白質 (xi)序列説明：SEQ ID NO ·· 125 :Gin Val Gin Ser Val Lys lie Ser Cys Leu 395 400 GCTGTTTCCT CACAATTGGC GAGATCCCAT GGATGATAA 1355 (2) Information of SEQ ID NO: 125: (i) Sequence characteristics: (A) Length: 401 Amino acid (B) Category: Amine Acid (D) topology: linear (ii) molecular class: protein (xi) sequence description: SEQ ID NO · · 125:

Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie 1 5 10 15Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie 1 5 10 15

Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30

Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45

Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60

Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80

Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu 85 90 95Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu 85 90 95

Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 ___-181 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 「裝·Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 ___- 181 This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling in this (Page)

、1T 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（179)1T 1221482 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of Invention (179)

Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125

Gly Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140Gly Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140

Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160

Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys 165 170 175Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys 165 170 175

Gly Asn Ala Thr His Asp Asn lie Cys Ser Gly Asn Ser Glu Ser Thr 180 185 190Gly Asn Ala Thr His Asp Asn lie Cys Ser Gly Asn Ser Glu Ser Thr 180 185 190

Gin Lys Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205Gin Lys Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205

Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 215 220Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 215 220

Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240

Lys Arg Gin His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255Lys Arg Gin His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255

Trp Lys His Gin Asn Lys Ala Gin Asp lie Val Lys Lys lie lie Gin 260 265 270Trp Lys His Gin Asn Lys Ala Gin Asp lie Val Lys Lys lie lie Gin 260 265 270

Asp lie Asp Leu Cys Glu Asn Ser Val Gin Arg His lie Gly His Ala 275 280 285Asp lie Asp Leu Cys Glu Asn Ser Val Gin Arg His lie Gly His Ala 275 280 285

Asn Leu Thr Phe Glu Gin Leu Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300Asn Leu Thr Phe Glu Gin Leu Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300

Lys Lys Val Gly Ala Glu Asp lie Glu Lys Thr lie Lys Ala Cys Lys 305 310 315 · 320Lys Lys Val Gly Ala Glu Asp lie Glu Lys Thr lie Lys Ala Cys Lys 305 310 315320

Pro Ser Asp Gin lie Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335Pro Ser Asp Gin lie Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335

Gly Asp Gin Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser 340 345 350Gly Asp Gin Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser 340 345 350

Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gin Ser Leu Lys Lys Thr 355 360 365 lie Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leu 370 375 380 -182- 本紙張尺度適用中國國家標準（CNS ) A4規格（210Χ297公釐） (請先閱讀背面之注意事項再填寫本頁) -裝·Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gin Ser Leu Lys Lys Thr 355 360 365 lie Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leu 370 375 380 -182- This paper is in accordance with Chinese national standards (CNS ) A4 size (210 × 297 mm) (Please read the precautions on the back before filling this page)

、1T 2 8 4 ix 2 12 A7 ______B7五、發明説明（180) 經濟部中央標準局員工消費合作社印製、 1T 2 8 4 ix 2 12 A7 ______B7 V. Description of the invention (180) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys 385 390 395 400 Leu (2) SEQ ID NO : 126的資訊： (i) 序列特性·· (A) 長度：139胺基酸 (B) 類別··胺基酸 (C) 股型··單股 (D) 拓樸學：線型 (ii) 分子類別··蛋白質 (xi)序列説明·· SEQ ID NO : 126 : Cys Pro Gin Gly Lys Tyr lie His Pro Gin Asn Asn Ser lie Cys Cys 15 10 15 Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro 20 25 30 Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala 35 40 45 Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys 50 55 60 Glu Met Gly Gin Val Glu lie Ser Ser Cys Thr Val Asp Arg Asp Thr 65 70 75 80 Val Cys Gly Cys Arg Lys Asn Gin Tyr Arg His Tyr Trp Ser Glu Asn 85 90 95 Leu Phe Gin Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His 100 105 110 Leu Ser Cys Gin Glu Lys Gin Asn Thr Val Cys Thr Cys His Ala Gly 115 120 125 Phe Phe Leu Arg Glu Asn Glu Cys Val Ser Cys 130 135 (請先閲讀背面之注意事項再填寫本頁) -裝. 訂 -183- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（181) (2) SEQ ID NO · 127的資訊： (i) 序列特性： (A) 長度：48鹼對！ (B) 類別「：核酸、 (C) 股型：單股 (D) 拓樸學：線型Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys 385 390 395 400 Leu (2) Information of SEQ ID NO: 126: (i) Sequence characteristics · (A) Length: 139 amino acid (B ) Category · Amino acid (C) Strand type · Single strand (D) Topology: Linear (ii) Molecular type · Protein (xi) sequence description · SEQ ID NO: 126: Cys Pro Gin Gly Lys Tyr lie His Pro Gin Asn Asn Ser lie Cys Cys 15 10 15 Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro 20 25 30 Gly Gin Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala 35 40 45 Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys 50 55 60 Glu Met Gly Gin Val Glu lie Ser Ser Cys Thr Val Asp Arg Asp Thr 65 70 75 80 Val Cys Gly Cys Arg Lys Asn Gin Tyr Arg His Tyr Trp Ser Glu Asn 85 90 95 Leu Phe Gin Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His 100 105 110 Leu Ser Cys Gin Glu Lys Gin Asn Thr Val Cys Thr Cys His Ala Gly 115 120 125 Phe Phe Leu Arg Glu Asn Glu Cys Val Ser Cys 130 135 (Please read the notes on the back before filling this page)- ORDER-183- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A7 B7 V. Description of the invention (181) (2) Information of SEQ ID NO · 127: (i) Sequence characteristics: ( A) Length: 48 alkali pairs! (B) Category ": Nucleic acid, (C) Stock type: Single strand (D) Topology: Linear

(ii) 分子類別」cDNA (xi)序列説明：SEQ ID NO : 127 : CCGGCGGACA TTTATCACAC AGCAGCTGAT GAGAAGTTTC TTCATCCA 48 (2) SEQ ID NO : 128的資訊·· (i) 序列特性： (A) 長度：219胺基酸 (B) 類別：胺基酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：蛋白質 (xi)序列説明：SEQ ID NO : 128 :(ii) Molecular type "cDNA (xi) Sequence description: SEQ ID NO: 127: CCGGCGGACA TTTATCACAC AGCAGCTGAT GAGAAGTTTC TTCATCCA 48 (2) Information of SEQ ID NO: 128 (i) Sequence characteristics: (A) Length: 219 amine Basic acid (B) Category: Amino acid (C) Strand type: Single strand (D) Topology: Linear (ii) Molecular type: Protein (xi) Sequence description: SEQ ID NO: 128:

Met Leu Gly lie Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala 15 10 15 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁)Met Leu Gly lie Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala 15 10 15 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the notes on the back before filling out this page)

Arg Leu Ser Ser Lys Ser Val Asn Ala Gin Val Thr Asp lie Asn Ser 20 25 30Arg Leu Ser Ser Lys Ser Val Asn Ala Gin Val Thr Asp lie Asn Ser 20 25 30

Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gin Asn 35 40 45Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gin Asn 35 40 45

Leu Glu Gly Leu His His Asp Gly Gin Phe Cys His Lys Pro Cys Pro 50 55 60Leu Glu Gly Leu His His Asp Gly Gin Phe Cys His Lys Pro Cys Pro 50 55 60

Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro 65 70 75 80 _-184- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐) 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（182)Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro 65 70 75 80 _-184- This paper size applies to China National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Printed by Consumer Cooperatives V. Invention Description (182)

Asp Cys Val Pro Cys Gin Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95Asp Cys Val Pro Cys Gin Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95

Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110

Leu Glu Val Glu lie Asn Cys Thr Arg Thr Gin Asn Thr Lys Cys Arg 115 120 125Leu Glu Val Glu lie Asn Cys Thr Arg Thr Gin Asn Thr Lys Cys Arg 115 120 125

Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 130 135 140Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 130 135 140

Pro Cys Thr Lys Cys Glu His Gly lie lie Lys Glu Cys Thr Leu Thr 145 150 155 160Pro Cys Thr Lys Cys Glu His Gly lie lie Lys Glu Cys Thr Leu Thr 145 150 155 160

Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Tr*p 165 170 175Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Tr * p 165 170 175

Leu Cys Leu Leu Leu Leu Pro He Pro Leu lie Val Trp Val Lys Arg 180 185 190Leu Cys Leu Leu Leu Leu Pro He Pro Leu lie Val Trp Val Lys Arg 180 185 190

Lys Glu Val Gin Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gin Gly 195 200 205Lys Glu Val Gin Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gin Gly 195 200 205

Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr 210 215 (2) SEQ ID NO · 129的資訊： (i) 序列特性： (A) 長度：280胺基酸 (B) 類別：胺基酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：蛋今質 (xi)序列説明：SEQ ID NO : 129 :Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr 210 215 (2) Information of SEQ ID NO · 129: (i) Sequence characteristics: (A) Length: 280 Amino acid (B) Category: Amino acid (C) Strand type: single strand (D) Topology: linear type (ii) Molecular type: egg present quality (xi) Sequence description: SEQ ID NO: 129:

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu 15 10 15Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu 15 10 15

Glu Leu Leu Val Gly lie Tyr Pro Ser Gly Val lie Gly Leu Val Pro 20 25 30Glu Leu Leu Val Gly lie Tyr Pro Ser Gly Val lie Gly Leu Val Pro 20 25 30

His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gin Gly Lys 35 40 45 185-His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gin Gly Lys 35 40 45 185-

本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐I (請先閲讀背面之注意事項再填寫本頁) -装·This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 mm I (Please read the precautions on the back before filling this page)-Pack ·

、1T 1221482 A7 B7 發明説明（183), 1T 1221482 A7 B7 Invention description (183)

Tyr lie His Pro Gin Asn Asn Ser lie Cys Cya Thr Lys Cys His Lys 50 55 60 Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gin Asp Thr Asp 65 70 75 80 Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu 85 90 95 Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gin Val 100 105 110 Glu lie Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg 115 120 125 Lys Asn Gin Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gin Cys Phe 130 135 140 Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gin Glu 145 150 155 160 Lys Gin Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu 165 170 175 (請先閲讀背面之注意事項再填寫本頁) * mV In β^—i ϋ— 一裝· 訂經濟部中央標準局員工消費合作社印製Tyr lie His Pro Gin Asn Asn Ser lie Cys Cya Thr Lys Cys His Lys 50 55 60 Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gin Asp Thr Asp 65 70 75 80 Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu 85 90 95 Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gin Val 100 105 110 Glu lie Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg 115 120 125 Lys Asn Gin Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gin Cys Phe 130 135 140 Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gin Glu 145 150 155 160 Lys Gin Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu 165 170 175 (Please read the notes on the back before filling out this page) * mV In β ^ —i ϋ— One pack · Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr 180 185 190 Lys Leu Cys Leu Pro Gin lie Glu Asn Val Lys Gly Thr Glu Asp Ser 195 200 205 Gly Thr Thr Val Leu Leu Pro Leu Val lie Phe Phe Gly Leu Cys Leu 210 215 220 Leu Ser Leu Leu Phe lie Gly Leu Met Tyr Arg Tyr Gin Arg Trp Lys 225 230 235 240 Ser Lys Leu Tyr Ser lie Val Cys Gly Lys Ser Thr Pro Glu Lys Glu 245 250 255Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr 180 185 190 Lys Leu Cys Leu Pro Gin lie Glu Asn Val Lys Gly Thr Glu Asp Ser 195 200 205 Gly Thr Thr Val Leu Leu Pro Leu Val lie Phe Phe Gly Leu Cys Leu 210 215 220 Leu Ser Leu Leu Phe lie Gly Leu Met Tyr Arg Tyr Gin Arg Trp Lys 225 230 235 240 Ser Lys Leu Tyr Ser lie Val Cys Gly Lys Ser Thr Pro Glu Lys Glu 245 250 255

Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser 260 265 270Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser 260 265 270

Phe Ser Pro Thr Pro Gly Phe Thr 275 280 -186 本紙張尺度適用中國國家標準（CNS ) A4規格（21 〇X 297公釐） 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（184) (2) SEQ ID NO : 130的資訊： (i) 序列特性： (A) 長度：207胺基酸 (B) 類別」胺基酸 (C) 股癞：單股 (D) 拓樸學：線型 (ii) 分子類別：蛋白質 (xi)序列説明：SEQ ID NO : 130 :Phe Ser Pro Thr Pro Gly Phe Thr 275 280 -186 This paper size is applicable to Chinese National Standard (CNS) A4 (21 × 297 mm) 1221482 A7 B7 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 184) (2) Information of SEQ ID NO: 130: (i) Sequence characteristics: (A) Length: 207 Amino acid (B) Category "Amino acid (C) Stocks: Single strand (D) Topology: Linear (ii) Molecular class: Protein (xi) Sequence description: SEQ ID NO: 130:

Met Leu Arg Leu lie Ala Leu Leu Val Cys Val Val Tyr Val Tyr Gly 1 5 10 15Met Leu Arg Leu lie Ala Leu Leu Val Cys Val Val Tyr Val Tyr Gly 1 5 10 15

Asp Asp Val Pro Tyr Ser Ser Asn Gin Gly Lys Cys Gly Gly His Asp 20 25 30Asp Asp Val Pro Tyr Ser Ser Asn Gin Gly Lys Cys Gly Gly His Asp 20 25 30

Tyr Glu Lys Asp Gly Leu Cys Cys Ala Ser Cys His Pro Gly Phe Tyr 35 40 45Tyr Glu Lys Asp Gly Leu Cys Cys Ala Ser Cys His Pro Gly Phe Tyr 35 40 45

Ala Ser Arg Leu Cys Gly Pro Gly Ser Asn Thr Val Cys Ser Pro Cys 50 55 60Ala Ser Arg Leu Cys Gly Pro Gly Ser Asn Thr Val Cys Ser Pro Cys 50 55 60

Glu Asp Gly Thr Phe Thr Ala Ser Thr Asn His Ala Pro Ala Cys Val 65 70 75 80Glu Asp Gly Thr Phe Thr Ala Ser Thr Asn His Ala Pro Ala Cys Val 65 70 75 80

Ser Cys Arg Gly Pro Cys Thr Gly His Leu Ser Glu Ser Gin Pro Cys 85 90 95Ser Cys Arg Gly Pro Cys Thr Gly His Leu Ser Glu Ser Gin Pro Cys 85 90 95

Asp Arg Thr His Asp Arg Val Cys Asn Cys Ser Thr Gly Asn Tyr Cys 100 105 110Asp Arg Thr His Asp Arg Val Cys Asn Cys Ser Thr Gly Asn Tyr Cys 100 105 110

Leu Leu Lys Gly Gin Asn Gly Cys Arg lie Cys Ala Pro Gin Thr Lys 115 120 125Leu Leu Lys Gly Gin Asn Gly Cys Arg lie Cys Ala Pro Gin Thr Lys 115 120 125

Cys Pro Ala Gly Tyr Gly Val Ser Gly His Thr Arg Ala Gly Asp Thr 130 135 140Cys Pro Ala Gly Tyr Gly Val Ser Gly His Thr Arg Ala Gly Asp Thr 130 135 140

Leu Cys Glu Lys Cys Pro Pro His Thr Tyr Ser Asp Ser Leu Ser Pro 145 150 155 160Leu Cys Glu Lys Cys Pro Pro His Thr Tyr Ser Asp Ser Leu Ser Pro 145 150 155 160

Thr Glu Arg Cys Gly Thr Ser Phe Asn Tyr lie Ser Val Gly Phe Asn 165 170 175Thr Glu Arg Cys Gly Thr Ser Phe Asn Tyr lie Ser Val Gly Phe Asn 165 170 175

Leu Tyr Pro Val Asn Glu Thr Ser Cys Thr Thr Thr Ala Gly His Asn 180 185 190 -187- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） ---------W裝-- (請先閲讀背面之注意事項再填寫本頁) 訂經濟部中央標準局員工消費合作社印製 1221482 A7 A7 B7 五、發明説明（18δ)Leu Tyr Pro Val Asn Glu Thr Ser Cys Thr Thr Thr Ala Gly His Asn 180 185 190 -187- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) --------- W Equipment-(Please read the precautions on the back before filling out this page) Order printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 1221482 A7 A7 B7 V. Description of the invention (18δ)

Glu Val lie Lys Thr Lys Glu Phe Thr Val Thr Leu Aen Tyr Thr 195 200 205 (2) SEQ ID NO ·· 131 的資訊： (i) 序列特性： (A) 長度：227胺基酸 (B) 類別：胺基酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：蛋白質 (xi)序列説明：SEQ ID NO : 131 :Glu Val lie Lys Thr Lys Glu Phe Thr Val Thr Leu Aen Tyr Thr 195 200 205 (2) Information of SEQ ID NO · · 131: (i) Sequence characteristics: (A) Length: 227 Amino acid (B) Category: Amino acid (C) strand type: single strand (D) topology: linear type (ii) molecular class: protein (xi) sequence description: SEQ ID NO: 131:

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu 15 10 15Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu 15 10 15

Trp Ala Ala Ala His Ala Leu Pro Ala Gin Val Ala Phe Thr Pro Tyr 20 25 30Trp Ala Ala Ala His Ala Leu Pro Ala Gin Val Ala Phe Thr Pro Tyr 20 25 30

Ala Pro Glu Pro Gly Ser Thr； Cys Arg Leu Arg Glu Tyr Tyr Asp Gin 35 40 45Ala Pro Glu Pro Gly Ser Thr; Cys Arg Leu Arg Glu Tyr Tyr Asp Gin 35 40 45

Thr Ala Gin Met Cys Cys Ser Lys Cys Ser Pro Gly Gin His Ala Lys 50 55 60Thr Ala Gin Met Cys Cys Ser Lys Cys Ser Pro Gly Gin His Ala Lys 50 55 60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 65 70 75 80Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 65 70 75 80

Ser Thr Tyr Thr Gin Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 85 90 95Ser Thr Tyr Thr Gin Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 85 90 95

Gly Ser Arg Cys Ser Ser Asp Gin Val Glu Thr Gin Ala Cys Thr Arg 100 105 110Gly Ser Arg Cys Ser Ser Asp Gin Val Glu Thr Gin Ala Cys Thr Arg 100 105 110

Glu Gin Asn Arg lie Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115 120 125Glu Gin Asn Arg lie Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115 120 125

Ser Lys Gin Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg !3〇 135 140Ser Lys Gin Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg! 3〇 135 140

Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val 145 150 155 160Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val 145 150 155 160

Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 165 170 175 -188- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁)Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 165 170 175 -188- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling in this page)

1221482 A7B7 經濟部中央標準局員工消費合作社印製五、發明説明（186)1221482 A7B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the Invention (186)

Asp He Cys Arg Pro His Gin He Cys Asn Val Val Ala He Pro Gly 180 185 190Asp He Cys Arg Pro His Gin He Cys Asn Val Val Ala He Pro Gly 180 185 190

Asn Ala Ser Arg Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200 205Asn Ala Ser Arg Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200 205

Met Ala Pro Gly Ala Val His Leu Pro Gin Pro Val Ser Thr Arg Ser 210 215 220Met Ala Pro Gly Ala Val His Leu Pro Gin Pro Val Ser Thr Arg Ser 210 215 220

Gin His Thr 225 (2) SEQ ID NO : 132的資訊： (i) 序列特性： (A) 長度：197胺基酸 · (B) 類別：胺基酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別··蛋白質 (xi)序列説明：SEQ ID NO : 132 :Gin His Thr 225 (2) Information of SEQ ID NO: 132: (i) Sequence characteristics: (A) Length: 197 amino acids (B) Category: amino acids (C) Strand type: single strand (D) Topology: linear (ii) molecular type · protein (xi) sequence description: SEQ ID NO: 132:

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr 1 5 10 15Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr 1 5 10 15

Ala Val His Leu Gly Gin Cys Val Thr Cys Ser Asp Lya Gin Tyr Leu 20 25 30 ^Ala Val His Leu Gly Gin Cys Val Thr Cys Ser Asp Lya Gin Tyr Leu 20 25 30 ^

His Asp Gly Gin Cys Cys Asp Leu Cys Gin Pro Gly Ser Arg Leu Thr 35 40 45His Asp Gly Gin Cys Cys Asp Leu Cys Gin Pro Gly Ser Arg Leu Thr 35 40 45

Ser His Cys Thr Ala Leu Glu Lys Thr Gin Cys His Pro Cys Asp Ser 50 55 60Ser His Cys Thr Ala Leu Glu Lys Thr Gin Cys His Pro Cys Asp Ser 50 55 60

Gly Glu Phe Ser Ala Gin Trp Asn Arg Glu lie Arg Cys His Gin His 65 70 75 80Gly Glu Phe Ser Ala Gin Trp Asn Arg Glu lie Arg Cys His Gin His 65 70 75 80

Arg His Cys Glu Pro Asn Gin Gly Leu Arg Val Lys Lys Glu Gly Thr 85 90 95Arg His Cys Glu Pro Asn Gin Gly Leu Arg Val Lys Lys Glu Gly Thr 85 90 95

Ala Glu Ser Asp Thr Val Cys Thr Cys Lys Glu Gly Gin His Cys Thr 100 105 110Ala Glu Ser Asp Thr Val Cys Thr Cys Lys Glu Gly Gin His Cys Thr 100 105 110

Ser Lys Asp Cys Glu Ala Cys Ala Gin His Thr Pro Cys lie Pro Gly 115 120 125 -189- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（187)Ser Lys Asp Cys Glu Ala Cys Ala Gin His Thr Pro Cys lie Pro Gly 115 120 125 -189- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling in this Page) Order 1221482 Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Description of Invention (187)

Phe Gly Val Met Glu Met Ala Thr Glu Thr Thr Asp Thr Val Cys His 130 135 140Phe Gly Val Met Glu Met Ala Thr Glu Thr Thr Asp Thr Val Cys His 130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Gin Ser Ser Leu Phe Glu Lys 145 150 155 160Pro Cys Pro Val Gly Phe Phe Ser Asn Gin Ser Ser Leu Phe Glu Lys 145 150 155 160

Cys Tyr Pro Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Val Leu Gin 165 170 175Cys Tyr Pro Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Val Leu Gin 165 170 175

Lys Gly Thr Ser Gin Thr Asn Val lie Cys Gly Leu Lys Ser Arg Met 180 185 190Lys Gly Thr Ser Gin Thr Asn Val lie Cys Gly Leu Lys Ser Arg Met 180 185 190

Arg Ala Leu Leu Val 195 (2) SEQ ID NO : 133的資訊： · (i) 序列特性： (A) 長度：208胺基酸 (B) 類Mi胺基酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：蛋白質 (xi)序列説明：SEQ ID NO : 133 :Arg Ala Leu Leu Val 195 (2) Information of SEQ ID NO: 133: · (i) Sequence characteristics: (A) Length: 208 amino acids (B) Mi-type amino acids (C) Share type: single strand ( D) Topology: Linear (ii) Molecular class: Protein (xi) Sequence description: SEQ ID NO: 133:

Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Phe Leu Asp lie lie 1 5 10 15Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Phe Leu Asp lie lie 1 5 10 15

Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30

Cys Pro Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Gys 65 70 75 80Cys Pro Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Gys 65 70 75 80

Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Thr Val Lys Gin Glu 85 90 95 _-190- _ 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) -裝· 訂 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（188)Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Thr Val Lys Gin Glu 85 90 95 _-190- _ This paper size applies to China National Standard (CNS) A4 size (210X297 mm) (Please read the precautions on the back before (Fill in this page)-Binding · 1221482 A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs V. Invention Description (188)

Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu lie Gin Lys 165 170 175Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu lie Gin Lys 165 170 175

Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 (2) SEQ ID NO : 134的資訊： ⑴序列特性： (A) 長度：224胺基酸 (B) 類別：胺基鹼 (C) 股型：單股 (D) 拓樸學：線型 (ii)分子類別··蛋白質 (xi)序列説明：8£(^10 1^0:134 :Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 (2) Information of SEQ ID NO: 134: ⑴ Sequence characteristics: (A) Length: 224 Amino acid (B) Category: Amine Alkali (C) strand type: single strand (D) topology: linear (ii) molecular type · protein (xi) sequence description: 8 £ (^ 10 1 ^ 0: 134:

Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu 15 10 15Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu 15 10 15

Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys 20 25 30Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys 20 25 30

Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn 35 40 45Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn 35 40 45

Leu Gly Glu Gly Val Ala Gin Pro Cys Gly Ala Asn Gin Thr Val Cys 50 55 60 -191 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X：Z97公釐） (請先閱讀背面之注意事項再填寫本頁) -裝· 訂 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（189)Leu Gly Glu Gly Val Ala Gin Pro Cys Gly Ala Asn Gin Thr Val Cys 50 55 60 -191-This paper size applies to China National Standard (CNS) A4 (210X: Z97 mm) (Please read the precautions on the back before (Fill in this page)-Binding · 1221482 A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs V. Invention Description (189)

Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr 65 70 75 80Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr 65 70 75 80

Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gin Ser Met Ser 85 90 95Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gin Ser Met Ser 85 90 95

Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly 100 105 110Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly 100 105 110

Tyr Tyr Gin Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys 115 120 125Tyr Tyr Gin Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys 115 120 125

Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gin Asp Lys Gin Asn Thr 130 135 140Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gin Asp Lys Gin Asn Thr 130 135 140

Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His 145 150 155 160Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His 145 150 155 160

Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gin 165 170 175Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gin 165 170 175

Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu lie Pro 180 185 190Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu lie Pro 180 185 190

Gly Arg Trp lie Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr 195 200 205Gly Arg Trp lie Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr 195 200 205

Ala Pro Ser Thr Gin Glu Pro Glu Ala Pro Pro Glu Gin Asp Leu lie 210 215 220 (2) SEQ ID NO : 135的資訊： (i) 序列特性： (A) 長度：2〇5胺基酸 (B) 類別：胺基酸 (C) 股瘛：單股 (D) 拓樸學：線型 (ii) 分子類別：蛋白質 (xi)序列説明：SEQ ID NO : 135 :Ala Pro Ser Thr Gin Glu Pro Glu Ala Pro Pro Glu Gin Asp Leu lie 210 215 220 (2) Information of SEQ ID NO: 135: (i) Sequence characteristics: (A) Length: 20.5 Amino acid (B) Class: Amino acid (C) Stock: Single strand (D) Topology: Linear (ii) Molecular class: Protein (xi) Sequence description: SEQ ID NO: 135:

Met Tyr Val Trp Val Gin Gin Pro Thr Ala Phe Leu Leu Leu Gly Leu 1 5 10 15Met Tyr Val Trp Val Gin Gin Pro Thr Ala Phe Leu Leu Leu Gly Leu 1 5 10 15

Ser Leu Gly Val Thr Val Lys Leu Asn Cys Val Lys Asp Thr Tyr Pro 20 25 30 -192- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝_ 、?τ 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（190)Ser Leu Gly Val Thr Val Lys Leu Asn Cys Val Lys Asp Thr Tyr Pro 20 25 30 -192- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) (Please read the precautions on the back before filling (This page) Equipment _,? Τ 1221482 A7 B7 Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs V. Invention Description (190)

Ser Gly His Lys Cys Cys Arg Glu Cys Gin Pro Gly His Gly Met Val 35 40 45Ser Gly His Lys Cys Cys Arg Glu Cys Gin Pro Gly His Gly Met Val 35 40 45

Ser Arg Cys Asp His Thr Arg Asp Thr Val Cys His Pro Cys Glu Pro 50 55 60Ser Arg Cys Asp His Thr Arg Asp Thr Val Cys His Pro Cys Glu Pro 50 55 60

Gly Phe Tyr Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gin Cys Thr 65 70 75 80Gly Phe Tyr Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gin Cys Thr 65 70 75 80

Gin Cys Asn His Arg Ser Gly Ser Glu Leu Lys Gin Asn Cys Thr Pro 85 90 95Gin Cys Asn His Arg Ser Gly Ser Glu Leu Lys Gin Asn Cys Thr Pro 85 90 95

Thr Glu Asp Thr Val Cys Gin Cys Arg Pro Gly Thr Gin Pro Arg Gin 100 105 110Thr Glu Asp Thr Val Cys Gin Cys Arg Pro Gly Thr Gin Pro Arg Gin 100 105 110

Asp Ser Ser His Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro Gly 115 120 125Asp Ser Ser His Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro Gly 115 120 125

His Phe Ser Pro Gly Ser Asn Gin Ala Cys Lys Pro Trp Thr Asn Cys 130 135 140His Phe Ser Pro Gly Ser Asn Gin Ala Cys Lys Pro Trp Thr Asn Cys 130 135 140

Thr Leu Ser Gly Lys Gin lie Arg His Pro Ala Ser Asn Ser Leu Asp 145 150 155 160Thr Leu Ser Gly Lys Gin lie Arg His Pro Ala Ser Asn Ser Leu Asp 145 150 155 160

Thr Val Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu Thr 165 170 175Thr Val Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu Thr 165 170 175

Gin Arg Thr Thr Phe Arg Pro Thr Thr Val Pro Ser Thr Thr Val Trp 180 185 190Gin Arg Thr Thr Phe Arg Pro Thr Thr Val Pro Ser Thr Thr Val Trp 180 185 190

Pro Arg Thr Ser Gin Leu Pro Ser Thr Pro Thr Leu Val 195 200 205 (2) SEQ ID NO : 136的資訊： (i) 序列特性： (A) 長度：191胺基酸 (B) 類別：胺基酸 (C) 股型：單股 (D) 拓樸學··線型 (ii) 分子類蚰：蛋白質 (xi)序列説明：SEQ ID NO ·· 136 :Pro Arg Thr Ser Gin Leu Pro Ser Thr Pro Thr Leu Val 195 200 205 (2) Information of SEQ ID NO: 136: (i) Sequence characteristics: (A) Length: 191 Amino acid (B) Category: Amino acid (C) Strand type: Single strand (D) Topology ·· Linear type (ii) Molecular type 蚰: Protein (xi) Sequence description: SEQ ID NO ·· 136:

Met Gly Asn Asn Cys Tyr Asn Val Val Val lie Val Leu Leu Leu Val 1 5 10 15 -193- 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) 裝· 訂 1221482 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（191)Met Gly Asn Asn Cys Tyr Asn Val Val Val lie Val Leu Leu Leu Val 1 5 10 15 -193- This paper size applies to the Chinese National Standard (CNS) A4 size (210 X 297 mm) (Please read the precautions on the back first (Fill in this page again) Binding and ordering 1221482 Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Invention Description (191)

Qly Cys Glu Lys Val Gly Ala Val Gin Asn Ser Cys Asp Asn Cys Gin 20 25 30Qly Cys Glu Lys Val Gly Ala Val Gin Asn Ser Cys Asp Asn Cys Gin 20 25 30

Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro 35 40 45Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro 35 40 45

Pro Ser Thr Phe Ser Ser lie Gly Gly Gin Pro Asn Cys Asn lie Cys 50 55 60Pro Ser Thr Phe Ser Ser lie Gly Gly Gin Pro Asn Cys Asn lie Cys 50 55 60

Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr 65 70 75 80Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr 65 70 75 80

His Asn Ala Glu Cys Glu Cys lie Glu Gly Phe His Cys Leu Gly Pro 85 90 95His Asn Ala Glu Cys Glu Cys lie Glu Gly Phe His Cys Leu Gly Pro 85 90 95

Gin Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gin Glu Leu Thr 100 105 110Gin Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gin Glu Leu Thr 100 105 110

Lys Gin Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gin Asn 115 120 125Lys Gin Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gin Asn 115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg 130 135 140Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg 130 135 140

Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro 145 150 155 160Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro 145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr lie Ser Val Thr Pro Glu 165 170 175Pro Val Val Ser Phe Ser Pro Ser Thr Thr lie Ser Val Thr Pro Glu 165 170 175

Gly Gly Pro Gly Gly His Ser Leu Gin Val Leu Thr Leu Phe Leu 180 185 190 (2) SEQ ID NO : 137的資訊： (i) 序列特性： (A) 長度：5 4驗對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型Gly Gly Pro Gly Gly His Ser Leu Gin Val Leu Thr Leu Phe Leu 180 185 190 (2) Information of SEQ ID NO: 137: (i) Sequence characteristics: (A) Length: 5 4 Verification pairs (B) Category: Nucleic acid (C) Stock type · Single stock (D) Topology: Linear

(ii) 分子類別·· cDNA (xi)序列説明：SEQ ID NO : 137 : TATGGATGAA GAAACTTCTC ATCAGCTGCT GTGTGATAAA TGTCCGCCGG GTAC 54 -194- 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝· 訂 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（192) (2) SEQ ID NO : 138的資訊： (i) 序列特性： (A) 長度·· 380胺基酸 (B) 類別：胺基酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：蛋白質〇ci)序列説明：SEQ ID NO : 138 :(ii) Molecular type · cDNA (xi) sequence description: SEQ ID NO: 137: TATGGATGAA GAAACTTCTC ATCAGCTGCT GTGTGATAAA TGTCCGCCGG GTAC 54 -194- This paper is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) Please read the notes on the back before filling this page) Binding and ordering 1221482 A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (192) (2) Information of SEQ ID NO: 138: Features: (A) Length · 380 Amino acid (B) Category: Amino acid (C) Strand type: Single strand (D) Topology: Linear type (ii) Molecular class: Protein 0ci) Sequence description: SEQ ID NO: 138:

Glu Thr Leu Pro Pro Lys Tyr Leu His Tyr Asp Pro Glu Thr Gly His 1 5 10 15 ^Glu Thr Leu Pro Pro Lys Tyr Leu His Tyr Asp Pro Glu Thr Gly His 1 5 10 15 ^

Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly Thr Tyr Leu Lys Gin His 20 25 30Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly Thr Tyr Leu Lys Gin His 20 25 30

Cys Thr Val Arg Arg Lys Thr Leu Cys Val Pro Cys Pro Asp His Ser 35 40 45Cys Thr Val Arg Arg Lys Thr Leu Cys Val Pro Cys Pro Asp His Ser 35 40 45

Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Val Tyr Cys Ser Pro 50 ; 55 60Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Val Tyr Cys Ser Pro 50; 55 60

Val Cys Lys Glu Leu Gin Ser Val Lys Gin Glu Cys Asn Arg Thr His 65 70 75 80Val Cys Lys Glu Leu Gin Ser Val Lys Gin Glu Cys Asn Arg Thr His 65 70 75 80

Asn Arg Val Cys Glu Cys Glu Glu Gly Arg Tyr Leu Glu lie Glu Phe 85 90 95Asn Arg Val Cys Glu Cys Glu Glu Gly Arg Tyr Leu Glu lie Glu Phe 85 90 95

Cys Leu Lys His Arg Ser Cys Pro Pro Gly Ser Gly Val Val Gin Ala 100 105 110Cys Leu Lys His Arg Ser Cys Pro Pro Gly Ser Gly Val Val Gin Ala 100 105 110

Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Lys Cys Pro Asp Gly Phe 115 120 125Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Lys Cys Pro Asp Gly Phe 115 120 125

Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys lie Lys His Thr Asn 130 135 140Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys lie Lys His Thr Asn 130 135 140

Cys Ser Thr Phe Gly Leu Leu Leu lie Gin Lys Gly Asn Ala Thr His 145 150 155 160Cys Ser Thr Phe Gly Leu Leu Leu lie Gin Lys Gly Asn Ala Thr His 145 150 155 160

Asp Asn Val Cys Ser Gly Asn Arg Glu Ala Thr Gin Lys Cys Gly lie 165 170 175Asp Asn Val Cys Ser Gly Asn Arg Glu Ala Thr Gin Lys Cys Gly lie 165 170 175

Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr 180 185 190 ___-195- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁) 訂 1221482 A7B7 經濟部中央標準局員工消費合作社印製五、發明説明（193)Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr 180 185 190 ___- 195- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the precautions on the back first (Fill in this page) Order 1221482 A7B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of Inventions (193)

Lys lie lie Pro Asn Trp Leu Ser Val Leu Val Asp Ser Leu Pro Gly 195 200 205Lys lie lie Pro Asn Trp Leu Ser Val Leu Val Asp Ser Leu Pro Gly 195 200 205

Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie Lys Arg Arg His Ser 210 215 220Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie Lys Arg Arg His Ser 210 215 220

Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn 225 230 235 * 240Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn 225 230 235 * 240

Arg Asp Gin Glu Met Val Lys Lys lie lie Gin Asp lie Asp Leu Cys 245 250 255Arg Asp Gin Glu Met Val Lys Lys lie lie Gin Asp lie Asp Leu Cys 245 250 255

Glu Ser Ser Val Gin Arg His Leu Gly His Ser Asn Leu Thr Thr Glu 260 265 270Glu Ser Ser Val Gin Arg His Leu Gly His Ser Asn Leu Thr Thr Glu 260 265 270

Gin Leu Leu Ala Leu Met Glu Ser Leu Pro Gly Lys Lys lie Ser Pro 275 280 285Gin Leu Leu Ala Leu Met Glu Ser Leu Pro Gly Lys Lys lie Ser Pro 275 280 285

Glu Glu lie Glu Arg Thr Arg Lys Thr Cys Lys Ser Ser Glu Gin Leu 290 295 300Glu Glu lie Glu Arg Thr Arg Lys Thr Cys Lys Ser Ser Glu Gin Leu 290 295 300

Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr 305 310 315 320Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr 305 310 315 320

Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu Lys Thr Ser His Phe 325 330 335Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu Lys Thr Ser His Phe 325 330 335

Pro Lys Thr Val Thr His Ser Leu Arg Lys Thr Met Arg Phe Leu His 340 345 350Pro Lys Thr Val Thr His Ser Leu Arg Lys Thr Met Arg Phe Leu His 340 345 350

Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu Phe Leu Glu Met lie 355 360 365Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu Phe Leu Glu Met lie 355 360 365

Gly Asn Gin Val Gin Ser Val Lys He Ser Cys Leu 370 375 380 (2) SEQ ID NO : 139的資訊： (i) 序列特性： (A) 長度：380胺基酸 (B) 類別：胺基酸 (c)股i :單股 (D)拓樸學：線型 (ii) 分子類別，蛋白質 -196- 本紙張尺度適用中國國家標準（CNS ) A4規格(210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝·Gly Asn Gin Val Gin Ser Val Lys He Ser Cys Leu 370 375 380 (2) Information of SEQ ID NO: 139: (i) Sequence characteristics: (A) Length: 380 Amino acid (B) Category: Amino acid ( c) Stock i: Single stock (D) Topology: Linear (ii) Molecular type, protein -196- This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before (Fill in this page)

、1T 1221482 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（194) (xi)序列説明：SEQ ID NO : 139 :1T 1221482 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (194) (xi) Sequence description: SEQ ID NO: 139:

Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His 15 10 15Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His 15 10 15

Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Ly3 Gin His 20 25 30Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Ly3 Gin His 20 25 30

Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr 35 40 45Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr 35 40 45

Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro 50 55 60Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro 50 55 60

Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu Cys Asn Arg Thr His 65 70 75 80Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu Cys Asn Arg Thr His 65 70 75 80

Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu lie Glu Phe 85 90 95Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu lie Glu Phe 85 90 95

Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gin Ala 100 105 110Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gin Ala 100 105 110

Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe 115 120 125Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe 115 120 125

Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn 130 135 140Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn 130 135 140

Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys Gly Asn Ala Thr His 145 150 155 160Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys Gly Asn Ala Thr His 145 150 155 160

Asp Asn lie Cys Ser Gly Asn Ser Glu Ser Thr Gin Lys Cys Gly lie 165 170 175Asp Asn lie Cys Ser Gly Asn Ser Glu Ser Thr Gin Lys Cys Gly lie 165 170 175

Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr 180 185 190Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr 180 185 190

Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly 195 200 205Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly 195 200 205

Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie Lys Arg Gin His Ser 210 215 220Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie Lys Arg Gin His Ser 210 215 220

Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn 225 230 235 240Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn 225 230 235 240

Lys Ala Gin Asp lie Val Lys Lys He lie Gin Asp lie Asp Leu Cys 245 250 255 -197- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐了 (請先閲讀背面之注意事項再填寫本頁) -裝. 訂 1221482 A7 B7 五、發明説明（195)Lys Ala Gin Asp lie Val Lys Lys He lie Gin Asp lie Asp Leu Cys 245 250 255 -197- This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm (please read the precautions on the back before filling in this Page)-Binding. Order 1221482 A7 B7 V. Description of Invention (195)

Glu Asn Ser Val Gin Arg His lie Gly His Ala Asn Leu Thr Phe Glu 260 265 270Glu Asn Ser Val Gin Arg His lie Gly His Ala Asn Leu Thr Phe Glu 260 265 270

Gin Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala 275 280 285 ^ Glu Asp lie Glu Lys Thr lie Lys Ala Cys Lys Pro Ser Asp Gin lie 290 295 300Gin Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala 275 280 285 ^ Glu Asp lie Glu Lys Thr lie Lys Ala Cys Lys Pro Ser Asp Gin lie 290 295 300

Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe 325 330 335Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe 325 330 335

Pro Lys Thr Val Thr Gin Ser Leu Lys Lys Thr lie Arg Phe Leu His 340 345 350Pro Lys Thr Val Thr Gin Ser Leu Lys Lys Thr lie Arg Phe Leu His 340 345 350

Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met lie 355 360 365Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met lie 355 360 365

Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys Leu 370 375 380 (2) SEQ ID NO : 140的資訊： (i) 序列特性： (A) 長度：30鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys Leu 370 375 380 (2) Information of SEQ ID NO: 140: (i) Sequence characteristics: (A) Length: 30 base pairs (B) Category: Nucleic acid (C) strand Type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 140 : TGGACCACCC AGAAGTACCT TCATTATGAC 、30 經濟部中央標準局員工消費合作社印製 (2) SEQ ID NO : 141的資訊： (i) 序列特性： (A) 長度：30鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 140: TGGACCACCC AGAAGTACCT TCATTATGAC, 30 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (2) Information of SEQ ID NO: 141: (i) Sequence characteristics : (A) Length: 30 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA • 198- 本紙張尺度適用中國,家標準（CNS ) A4規格（210 X 297公釐） 1221482 A7 B7 五、發明説明（196) (xi)序列説明：SEQ ID NO : 141 : GTCATAATGA AGGTACTTCT GGGTGGTCCA 30 (2) SEQ ID NO ·· 142的資訊： (i) 序列特性： (A) 長度：3 1鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA • 198- This paper is in Chinese standard (CNS) A4 size (210 X 297 mm) 1221482 A7 B7 V. Description of the invention (196) (xi) Sequence description: SEQ ID NO: 141: GTCATAATGA AGGTACTTCT GGGTGGTCCA 30 (2) Information of SEQ ID NO · · 142: (i) Sequence characteristics: (A) Length: 3 1 base pairs (B) Category: nucleic acid (C) Share type: single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 142 : GGACCACCCA GCTTCATTAT GACGAAGAAA C 31 (2) SEQ ID NO : 143的資訊： (i) 序列特性： (A) 長度：3 1驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 142: GGACCACCCA GCTTCATTAT GACGAAGAAA C 31 (2) Information of SEQ ID NO: 143: (i) Sequence characteristics: (A) Length: 3 1 pair check (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別·· cDNA (xi)序列説明：SEQ ID NO : 143 : GTTTCTTCGT CATAATGAAG CTGGGTGGTC C 31 (2) SEQ ID NO : 144的資訊·· (i) 序列特性： (A) 長度：29鹼對 (B) 類別：核酸 (C) 股型：單股經濟部中央標準局員工消費合作社印製 (D) 拓樸學：線型(ii) Molecular type · cDNA (xi) Sequence description: SEQ ID NO: 143: GTTTCTTCGT CATAATGAAG CTGGGTGGTC C 31 (2) Information of SEQ ID NO: 144 (i) Sequence characteristics: (A) Length: 29 bases Pair (B) Category: Nucleic Acid (C) Stock Type: Single Stock Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 144 : GTGGACCACC CAGGACGAAG AAACCTCTC 29 (2) SEQ ID NO : 145的資訊： (i)序列特性： -199- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（197) (A) 長度：29鹼對 (B) 類別：核酸 (C) 股型：單股(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 144: GTGGACCACC CAGGACGAAG AAACCTCTC 29 (2) Information of SEQ ID NO: 145: (i) Sequence characteristics: -199- This paper size applies Chinese national standards (CNS) A4 specification (210X 297 mm) 1221482 A7 B7 V. Description of the invention (197) (A) Length: 29 base pairs (B) Category: Nucleic acid (C) Stock type: Single strand

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 145 : GAGAGGTTTC TTCGTCCTGG GTGGTCCAC 29 (2) SEQ ID NO : 146的資訊·· (i) 序列特性： (A) 長度：29鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 145: GAGAGGTTTC TTCGTCCTGG GTGGTCCAC 29 (2) Information of SEQ ID NO: 146 (i) Sequence characteristics: (A ) Length: 29 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 146 : CGTTTCCTCC AAAGTTCCTT CATTATGAC 29 (2) SEQ ID NO : 147的資訊： (i) 序列特性： (A) 長度：29鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 146: CGTTTCCTCC AAAGTTCCTT CATTATGAC 29 (2) Information of SEQ ID NO: 147: (i) Sequence characteristics: (A) Length: 29 base pairs (B ) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 147 : GTCATAATGA AGGAACTTTG GAGGAAACG 29 經濟部中央標準局員工消費合作社印製 (2) SEQ ID NO ·· 148的資訊： (i) 序列特性： (A) 長度：32鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 147: GTCATAATGA AGGAACTTTG GAGGAAACG 29 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (2) Information of SEQ ID NO · · 148: (i) Sequence characteristics : (A) Length: 32 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分予類別：cDNA -200- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1221482 A7 B7 五、發明説明（198) (xi)序列説明：SEQ ID NO : 148 : GGAAACGTTT CCTGCAAAGT ACCTTCATTA TG 32 (2) SEQ ID NO : 149的資訊： (i) 序列特性： (A) 長度：32鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Classification: cDNA -200- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) 1221482 A7 B7 V. Description of the invention (198) (xi) Sequence description: SEQ ID NO: 148: GGAAACGTTT CCTGCAAAGT ACCTTCATTA TG 32 (2) Information of SEQ ID NO: 149: (i) Sequence characteristics: (A) Length: 32 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 149 : CATAATGAAG GTACTTTGCA GGAAACGTTT C 32 (2) SEQ ID NO ·· 150的資訊： (i) 序列特性： (A) 長度：2 7驗對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 149: CATAATGAAG GTACTTTGCA GGAAACGTTT C 32 (2) Information of SEQ ID NO · · 150: (i) Sequence characteristics: (A) Length: 2 7 Pair (B) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 150 : CACGCAAAAG TCGGGAATAG ATGTCAC 27 (2) SEQ ID NO : 151 的資訊： (i) 序列特性： (A) 長度：27鹼對 (B) 類別：核酸 (C) 股型：單股經濟部中央標準局員工消費合作社印製 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 150: CACGCAAAAG TCGGGAATAG ATGTCAC 27 (2) Information of SEQ ID NO: 151: (i) Sequence characteristics: (A) Length: 27 base pairs (B ) Category: Nucleic Acid (C) Stock Type: Single Stock Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 151 : GTGACATCTA TTCCCGACTT TTGCGTG 27 (2) SEQ ID NO : 152的資訊·· (i)序列特性： -201 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（199) (A) 長度：25鹼對 (B) 類別：核酸 (C) 股型：單股(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 151: GTGACATCTA TTCCCGACTT TTGCGTG 27 (2) Information of SEQ ID NO: 152 (i) Sequence characteristics: -201-This paper is for Chinese countries Standard (CNS) A4 specification (210X 297 mm) 1221482 A7 B7 V. Description of the invention (199) (A) Length: 25 base pairs (B) Category: Nucleic acid (C) Stock type: single strand

(D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明·· SEQ ID NO ·· 152 : CACCCTGTCG GAAGAGGCCT TCTTC 25 (2) SEQ ID NO : 153的資訊： (i) 序列特性： (A) 長度：25鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: Linear (ii) Molecular category: cDNA (xi) Sequence description · SEQ ID NO · 152: CACCCTGTCG GAAGAGGCCT TCTTC 25 (2) Information of SEQ ID NO: 153: (i) Sequence characteristics: (i) A) Length: 25 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 153 : GAAGAAGGCC TCTTCCGACA GGGTG 25 (2) SEQ ID NO : 154的資訊： (i) 序列特性： (A) 長度：24鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 153: GAAGAAGGCC TCTTCCGACA GGGTG 25 (2) Information of SEQ ID NO: 154: (i) Sequence characteristics: (A) Length: 24 base pairs (B ) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 154 : TGACCTCTCG GAAAGCAGCG TGCA 24 經濟部中央標準局員工消費合作社印製 (2) SEQ ID NO : 155的資訊： (i) 序列特性： (A) 長度：24鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 154: TGACCTCTCG GAAAGCAGCG TGCA 24 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (2) Information of SEQ ID NO: 155: (i) Sequence characteristics: (A) Length: 24 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA -202- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1221482 A7 B7 五、發明説明（2〇〇) (xi)序列説明：SEQ ID NO : 155 : TGCACGCTGC TTTCCGAGAG GTCA 24 (2) SEQ ID NO : 156的資訊： (i) 序列特性： (A) 長度：24鹼對 (B) 類別··核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA -202- This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm) 1221482 A7 B7 V. Description of the invention (200) (xi) Sequence description: SEQ ID NO: 155: TGCACGCTGC TTTCCGAGAG GTCA 24 (2) Information of SEQ ID NO: 156: (i) Sequence characteristics: (A) Length: 24 base pairs (B) Category · Nucleic acid (C) Strand type: Single strand (D) Extension Park Xue: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 156 : CCTCGAAATC GAGCGAGCAG CTCC 24 (2) SEQ ID NO : 157的資訊： (i) 序列特性： (A) 長度：25鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 156: CCTCGAAATC GAGCGAGCAG CTCC 24 (2) Information of SEQ ID NO: 157: (i) Sequence characteristics: (A) Length: 25 base pairs (B ) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 157 : CGATTTCGAG GTCTTTCTCG TTCTC 25 (2) SEQ ID NO : 158的資訊： (i) 序列特性： (A) 長度：33鹼對 (B) 類別：核酸 (C) 股型··單股經濟部中央標準局員工消費合作社印製 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 157: CGATTTCGAG GTCTTTCTCG TTCTC 25 (2) Information of SEQ ID NO: 158: (i) Sequence characteristics: (A) Length: 33 base pairs (B ) Category: Nucleic acid (C) Stock type ·· Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 158 : CCGTGAAAAT AAGCTCGTTA TAACTAGGAA TGG 33 (2) SEQ ID NO ·· 159的資訊： (i)序列特性： -203- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 48 2 2 A7 B7 五、發明説明（2〇1) (A) 長度·· 3 3鹼對 (B) 類別：核酸 (請先閱讀背面之注意事項再填寫本頁) (C) 股型：單股(ii) Molecular category: cDNA (xi) Sequence description: SEQ ID NO: 158: CCGTGAAAAT AAGCTCGTTA TAACTAGGAA TGG 33 (2) Information of SEQ ID NO · · 159: (i) Sequence characteristics: -203- This paper is applicable to China National Standard (CNS) A4 Specification (210X 297 mm) 48 2 2 A7 B7 V. Description of the Invention (2) (A) Length · 3 3 Alkali Pair (B) Category: Nucleic Acid (Please read the notes on the back first (Please fill in this page for matters) (C) Share type: single share

(D) 拓樸學：線型 (ii)分子類別·· cDNA (xi)序列説明：SEQ ID NO : 159 : CCATTCCTAG TTATAACGAG CTTATTTTCA CGG 33 (2) SEQ ID NO : 160的資訊： (i) 序列特性： (A) 長度：3 8鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(D) Topology: linear (ii) molecular type · cDNA (xi) sequence description: SEQ ID NO: 159: CCATTCCTAG TTATAACGAG CTTATTTTCA CGG 33 (2) Information of SEQ ID NO: 160: (i) Sequence characteristics: (i) A) Length: 3 8 Base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO ·· 160 : CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) SEQ ID NO : 161 的資訊： (i) 序列特性： (A) 長度：44鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO. · 160: CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) Information of SEQ ID NO: 161: (i) Sequence characteristics: (A) Length: 44 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 161 : CCTCTCTCGA GTCAGGTGAC ATCTATTCCA CACTTTTGCG TGGC 44 經濟部中央標準局員工消費合作社印裝 (2) SEQ ID NO : 162的資訊： (i) 序列特性： (A) 長度：3 8鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 161: CCTCTCTCGA GTCAGGTGAC ATCTATTCCA CACTTTTGCG TGGC 44 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (2) Information of SEQ ID NO: 162: (i) Sequence Characteristics: (A) Length: 3 8 Base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA -204- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） π N 48 1± 2 12 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（2〇2) (xi)序列説明：SEQ ID NO : 162 : CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) SEQ ID NO : 163的資訊： (i) 序列特性： (A) 長度：38鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular type: cDNA -204- This paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) π N 48 1 ± 2 12 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (20.2) (xi) Sequence description: SEQ ID NO: 162: CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) Information of SEQ ID NO: 163: (i) Sequence characteristics: (A) Length: 38 base pairs (B) Category: Nucleic Acid (C) Strand Type: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 163 : CCTCTCTCGA GTCAAGGAAC AGCAAACCTG AAGAAGGC 38 (2) SEQ ID NO : 164的資訊： (i) 序列特性： (A) 長度：38鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 163: CCTCTCTCGA GTCAAGGAAC AGCAAACCTG AAGAAGGC 38 (2) Information of SEQ ID NO: 164: (i) Sequence characteristics: (A) Length: 38 base pairs ( B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 164 : CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) SEQ ID NO : 165的資訊： (i) 序列特性： (A) 長度：38鹼對 (B) 類別··核酸 (C) 股型：單股 (D) 拓樸學：線型(ii) Molecular class: cDNA (xi) Sequence description: SEQ ID NO: 164: CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) Information of SEQ ID NO: 165: (i) Sequence characteristics: (A) Length: 38 base pairs ( B) Category ·· Nucleic Acid (C) Strand: Single Strand (D) Topology: Linear

(ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 165 : CCTCTCTCGA GTCACTCTGT GGTGAGGTTC GAGTGGCC 38 (2) SEQ ID NO ·· 166的資訊·· (i)序列特性： -205- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) •裝· 訂 1221482 A7 B7五、發明説明（2〇3)(ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 165: CCTCTCTCGA GTCACTCTGT GGTGAGGTTC GAGTGGCC 38 (2) Information of SEQ ID NO · · 166 · (i) Sequence characteristics: -205- Applicable on this paper scale China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) • Binding and ordering 1221482 A7 B7 V. Description of the invention (203)

經濟部中央標準局員工消費合作社印I (A) 長度：3 8鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型 (ii)分子類別：cDNA (xi)序列説明：SEQ ID NO : 166 : CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) SEQ ID NO : 167的資訊： (i) 序列特性： (A) 長度：38鹼對 (B) 類別：核酸 (C) 股型··單股 (D) 拓樸學：線型 (ii) 分子類別：cDNA (xi)序列説明：SEQ ID NO : 167 : CCTCTCTCGA GTCAGGATGT TTTCAAGTGC TTGAGGGC 38 (2) SEQ ID NO : 168 的資;訊： (i) 序列特性： ’ (A) 長度：16鹼對 (B) 類別：核酸 (C) 股型：單股 (D) 拓樸學：線型 (ii) 分子類別：曼白質 (xi)序列説明：SEQ ID NO : 168 : Met Lys His His His His His His His Ala Ser Val Asn Ala Leu Glu 15 10 15 (請先閲讀背面之注意事項再填寫本頁) 裝· 訂 -206- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）Employees' Cooperative Cooperative I of the Central Bureau of Standards of the Ministry of Economic Affairs I (A) Length: 3 8 Alkali pairs (B) Category: Nucleic acid (C) Stock type: Single strand (D) Topology: Linear type (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 166: CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (2) Information of SEQ ID NO: 167: (i) Sequence characteristics: (A) Length: 38 base pairs (B) Category: Nucleic acid (C) Share type ·· Single strand (D) Topology: Linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 167: CCTCTCTCGA GTCAGGATGT TTTCAAGTGC TTGAGGGC 38 (2) Resources of SEQ ID NO: 168; News: (i ) Sequence characteristics: '(A) Length: 16 base pairs (B) Category: Nucleic acid (C) Strand type: Single strand (D) Topology: Linear type (ii) Molecular type: Mann white matter (xi) Sequence description: SEQ ID NO: 168: Met Lys His His His His His His His Ala Ser Val Asn Ala Leu Glu 15 10 15 (Please read the precautions on the back before filling out this page) Binding · Binding -206- This paper size applies to Chinese national standards ( CNS) A4 size (210X297 mm)

Claims

1221482 第086104638號專利申請案 B8 中文申請專利範圍替換本(93年6月）S 公告本六、申請專利範園胳/^1修正補充 1. 一種分離出的核酸，其編碼具有至少抑制骨重吸收活性之促骨堆積肽（〇 P G)之多肽’其中該核酸係選自下列所成組合之中者： a) SEQ ID NO : 120，SEQ ID NO : 122，和 SEQ ID NO : 124中所示核酸或其互補股； b) 可於4 2Ό下5X SSC、50%甲醯胺及0·5ο/〇 SDS之嚴格條件與 SEQ ID NO: 120、SEQ ID NO: 122 及 SEQ ID NO: 124所示之多肽編碼區之互補股雜合’並可於室溫下2X SSC、55°C下IX SSC及55°C下〇·5Χ S S C之連續沖洗條件下保持雜合狀態之核酸， SEQ ID NO: 120: ATCAAAGGCA GGGCATACTT CCTGTTGCCC AGACCTTATA TAAAACGTCA TGTTCGCCTG 60 GGCAGCAGAG AAGCACCTAG CACTGGCCCA GCGGCTGCCG CCTGAGGTTT CCAGAGGACC 120 ACA ATG AAC .AA^ TGG CTG TGC TGT GCA CTC CTG GTG TTC TTG GAC ATC 1S8 Met,Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Phe Leu Asp lie 1 5 10 15 ATT GAA TGG ACA ACC CAG GAA ACC TTT CCT CCA AAA TAC TTG CAT TAT 216 lie Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lvs Tyr Leu His Tyr ' 20 25 30 GAC CCA GAA ACC GGA CGT CAG CTC TTG TGT GAC AAA TGT GCT CCT GGC 2S4 Aap Pro Glu Thr Gly Arg Gin Leu Leu Cys Asp Lya Cys Ala Pro Gly 35 40 45 ACC TAC CTA AAA *CAG CAC TGC ACA GTC AGG AGG AAG ACA CTG TGT GTC 312 Thr Tyr Leu Lvs Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val 50 55 60 CCT TGC CCT GAC TAC TCT TAT ACA GAC AGC TGG CAC ACG AGT GAT GAA 360 Pro Cys Pro Aap Tyr Ser Tyr Thr Aap Ser Trp Hia Thr Ser A3p Glu S5 70 75 O:\45\45893-93062 l.DOC\su 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1221482 C8 D8 六、申請專利範園 TGC GTG TAC TGC AGC ccc GTG TGC AAG GAA CTG CAG ACC GTG ΛΑΑ CAG 408 Cys Val Tyr Cys Ser Pro Val Cys Lys. Glu Leu Gin Thr Val Lys Gin 80 85 90 95 GAG TGC AAC CGC ACC CAC AAC CGA GTG TGC GAA TGT GAG GAA GGG CGC 456 Glu Cys Asn Arg Thr His Asn Arg Val Cvs Glu Cys Glu Glu Gly Arg 100 105 1Ϊ0 TAC CTG GAG CTC GAA TTC TGC TTG AAG CAC CGG AGC TGT CCC GCA GGC 504 Tyr Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly 115 120 125 TTG GGT GTG CTG. -CAG GCT GGG ACC CCA GAG CGA AAC ACG GTT TGC AAA 552 Leu Gly Val Leu Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys T30 135 140 AGA TGT CCG GAT GGG TTC TTC TCA GGT GAG ACG TCA TCG AAA GCA CCC 600 Arg Cys Pro Asp'Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro 145 150 155 TGT AGG AAA CAC ACC AAC TGC AGC TCA CTT GGC CTC CTG CTA ATT CAG 64¾ Cys Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu lie Gin 160 165 170 175 AAA GGA GCA ACA CAT GAC AAT GTA TGT TCC GGA AAC AGA GAA GCA · 696 Lys Gly Asn Ala Thr His Asp Asn Val Cys Ser Gly Asn Arg Glu Ala 180 185 190 ACT CAA AAT TGT GGA ATA GAT GTC ACC CTG TGC GAA GAG GCA TTC TTC 744 Thr Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe * ' 195 200 205 AGG TTT GCT GTG CCT ACC AAG ATT ATA CCG AAT TGG CTG AGT GTT CTG 792 Arg Phe Ala Val Pro Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu 210 215 220 GTG GAC AGT TTG CCT GGG ACC AAA GTG AAT GCA GAG AGT GTA GAG AGG 840 Val Asp Ser Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg 225 230 235 ATA AAA CGG AGA CAC AGC TCG CAA GAG CAA ACT TTC CAG CTA CTT AAG 888 lie Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys 240 245 250 255 CTG TGG AAG CAT CAA AAC AGA GAC CAG GAA •ATG GTG AAG AAG ATC ATC 936 Leu Trp Lys His Gin Asn Arg Asp Gin Glu Met Val Lys Lys lie lie 260 265 270 CAA GAC ATT GAC CTC TGT GAA AGC AGT GTG CAA CGG CAT ATC GGC CAC 984 Gin Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His lie Gly His 275 280 285 • 2- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1221482 A8 B8 C8 D8 々、申請專利範圍 GCG AAC CTC ACC ACA GAG CAG CTC CGC ATC TTG ATG GAG AGC TTG CCT Ala Asn Leu Thr Thr Glu Gin Leu Arg'Ile Leu Met Glu Ser Leu Pro 290 295 300 GGG AAG AAG ATC AGC CCA GAC GAG ATT GAG AGA ACG AGA AAG ACC TGC Giy Lys Lys lie Ser Pro Asp Glu lie Glu Arg Thr Arg Lys Thr Cys 305 310 315 AAA CCC AGC GAG CAG CTC CTG AAG CTA CTG AGC TTG TGG AGG ATC AAA Lys Pro Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys 320 325 330 335 AAT GGA GAC CAA *GAC ACC TTG AAG GGC CTG ATG TAC GCA CTC AAG CAC Asn Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His 340 345 350 * i- TTG AAA GCA -TAC .CAC TTT CCC AAA ACC GTC ACC CAC AGT CTG AGG AAG Leu Lys Ala Tyr His Phe Pro Lys Thr Val Thr His Ser Leu Arg Lys 355 360 365 ACC ATC AGG TTC TTG CAC AGC TTC ACC ATG TAC CGA TTG TAT CAG AAA Thr lie Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys 370 375 380 CTC TTT CTA GAA ATG ATA GGG AAT CAG GTT CAA TCA GTG AAG ATA AGC Leu Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser 385 390 395 TGC TTA TAGTTAGGAA TGGTCACTGG GCTGTTTCTT CAGGATGGGC CAACACTGAT Cys Leu 400 GGAGCAGATG GCTGCTTCTC CGGCTCTTGA AATGGCAGTT GATTCCTTTC TCATCAGTTG GTGGGAATGA AGATCCTCCA GCCCAACACA CACACTGGGG AGTCTGAGTC AGGAGAGTGA GGCAGGCTAT TTGATAATTG TGCAAAGCTG CCAGGTGTAC ACCTAGAAAG TCAAGCACCC TGAGAAAGAG GATATTTTTA TAACCTCAAA CATAGGCCCT TTCCTTCCTC TCCTTATGGA TGAGTACTCA GAAGGCTTCT ACTATCTTCT GTGTCATCCC TAGATGAAGG CCTCTTTTAT TTATTTTTTT ATTCTTTTTT TCGGAGCTGG GGACCGAACC CAGGGCCTTG CGCTTGCGAG GCAAGTGCTC TACCACTGAG CTAAATCTCC AACCCCTGAA GGCCTCTTTC TTTCTGCCTC TGATAGTCTA TGACATTCTT TTTTCTACAA TTCGTATCAG GTGCACGAGC CTTATCCCAT TTGTAGGTTT CTAGGCAAGT TGACCGTTAG CTATTTTTCC CTCTGAAGAT TTGATTCGAG TTGCAGACTT GGCTAGACAA GCAGGGGTAG GTTATGGTAG TTTATTTAAC AGACTGCCAC -3- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1032 1080 1128 1176 1224 lil2 1320 1376 1436 1496 1556 1616 1676 1736 1796 1856 1916 1976 1221482 A8 B8 C8 D8 六、申請專利範圍 CAGGAGTCCA GTGTTTCTTG TTCCTCTGTA GTTGTACCTA AGCTGACTCC AAGTACATTT 2036 AGTATGAAAA ATAATCAACA AATTTTATTC CTTCTATCAA CATTGGCTAG CTTTGTTTCA 2096 GGGCACTAAA AGAAACTACT ATATGGAGAA AGAATTGATA TTGCCCCCAA CGTTCAACAA 2156 CCCAATAGTT TATCCAGCTG TCATGCCTGG TTCAGTGTCT ACTGACTATG CGCCCTCTTA 2216 TTACTGCATG CAGTAATTCA ACTGGAAATA GTAATAATAA TAATAGAAAT AAAATCTAGA 2276 CTCCATTGGA TCTC.TCTGAA TATGGGAATA TCTAACTTAA GAAGCTTTGA GATTTCAGTT 2336 GTGTTAAAGG CTTTTATTAA AAAGCTGATG CTCTTCTGTA AAAGTTACTA ATATATCTGT 2396 AAGACTATTA CAGTATTGCT ATTTATATCC ATCCAG 2432 SEQ ID NO: 122 CCTTATATAA ACGTCATGAT TGCCTGGGCT GCAGAGACGC ACCTAGCACT GACCCAGCGG * . · 60 CTGCCTCCTG AGGTTTCCCG AGGACCACA ATG AAC AAG Met Asn Lys 1 TGG CTG TGC Trp Leu Cys 5 TGC GCA Cys Ala 113 CTC CTG GTG CTC CTG GAC ATC ATT GAA TGG ACA ACC CAG GAA ACC CTT 161 Leu Leu Val Leu Leu Asp lie lie Glu Trp Thr Thr Gin Glu Thr Leu 10 15 20 CCT CCA AAG TAC TTG CAT TAT GAC CCA GAA ACT GGT CAT CAG CTC CTG 209 Pro Pro Lys Tyr Leu His Tyr Asp Pro Glu Thr Gly His Gin Leu Leu 25 30 35 40 TGT GAC AAA TGT GCT CCT GGC ACC TAC CTA AAA CAG CAC TGC ACA GTG 257 Cys Asp Lys Cys Ala Pro Gly Thr Tyr Leu Lys Gin Kis Cys Thr Val 45 50 55 AGG AGG AAG ACA TTG TGT GTC CCT TGC CCT GAC CAC TCT TAT ACG GAC 305 Arg Arg Lys Thr Leu Cys Val Pro Cys Pro Asp His Ser Tyr Thr Asp βα 65 * *'70 AGC TGG CAC ACC AGT GAT GAG TGT GTG TAT TGC AGC CCA GTG TGC AAG 353 Ser Trp His Thr Ser Asp Glu Cys Val Tyr Cys Ser Pro Val Cys Lys 75 80 85 -4- 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) 1221482 A8 B8 C8 D8 六、申請專利範圍 GAA CTG CAG TCC GTG AAG CAG GAG TGC AAC CGC ACC CAC AAC CGA GTG Glu Leu Gin Ser Val Lys Gin Glu Cys Asn Arg Thr His Asn Arg Val 90 95 100 TGT GAG TGT GAG GAA GGG CGT TAC CTG GAG ATC GAA TTC TGC TTG AAG Cys Glu Cys Glu Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu Lys 105 110 115 120 CAC CGG AGC TGT CCC CCG GGC TCC GGC GTG GTG CAA GCT GGA ACC CCA His Arg Ser Cys Pro Pro Gly Ser Gly Val Val Gin Ala Gly Thr Pro 125 130 135 GAG CGA· AAC ACA GTT TGC AAA AAA TGT CCA GAT GGG TTC TTC TCA GGT Glu Arg Asn"Thr-Val Cys Lys Lys Cys Pro Asp Gly Phe Phe Ser Gly 140 145 150 GAG ACT TCA TCG AAA GCA CCC TGT ATA AAA CAC ACG AAC TGC AGC ACA Glu Thr Ser Ser Lys Ala Pro Cys lie Lys His Thr Asn Cys Ser Thr 155 160 165 TTT GGC CTC CTG CTA ATT CAG AAA GGA AAT GCA ACA CAT GAC AAC GTG Phe Gly Leu Leu Leu lie Gin Lys Gly Asn Ala Thr His Asp Asn Val 17 0 175 180 TGT TCC GGA' AAC AGA GAA GCC ACG CAA AAG TGT GGA ATA GAT GTC ACC Cys Ser Gly Asn Arg Glu Ala Thr Gin Lys Cys Gly lie Asp Val Thr 185 190 195 200 CTG TGT GAA GAG GCC TTC TTC AGG TTT GCT GTT CCT ACC AAG ATT ATA Leu Cys Glu Glu Ala Phe Phe Arg Phe •Ala Val Pro Thr Lys lie lie 205 210 215 CCA AAT TGG CTG AGT GTT TTG GTG GAC AGT TTG CCT GGG ACC AAA GTG Pro Asn Trp Leu Ser Val Leu Val Asp Ser Leu Pro Gly Thr Lys Val 220 225 230 AAT GCC GAG AGT GTA GAG AGG ATA AAA CGG AGA CAC AGC TCA CAA GAG Asn Ala Glu Ser Val Glu Arg lie Lys Arg Arg His Ser Ser Gin Glu 235 240 245 CAA ACC TTC CAG CTG CTG AAG CTG TGG AAA CAT CAA ；AAC AGA GAC .CAG Gin Thr Phe Gin Leu Leu Lys Leu ,Trp Lys His Gin Asn Arg Asp Gin 250 255 260 GAA ATG GTG AAG AAG ATC ATC CAA GAC ATT GAC CTC TGT GAA AGC AGC Glu Met Val Lys Lys lie lie Gin Asp lie Asp Leu Cys Glu Ser Ser 265 27 0 275 280 401 449 497 545 593 641 689 73*7 785 833 881 929 -5-本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) 1221482 as Jd8 C8 D8 六、申請專利範圍 GTG CAG CGG CAT CTC GGC CAC TCG AAC CTC ACC ACA GAG CAG CTT CTT 977 Vai Gin Arg His Leu Gly His Ser Asn Leu Thr Thr Glu Gin Leu Leu 285 290 295 GCC TTG ATG GAG AGC CTG CCT GGG AAG AAG ATC AGC CCA GAA GAG ATT 1025 Ala Leu Met Glu Ser Leu Pro Gly Lys Lys lie Ser Pro Glu Glu lie 300 305 310 GAG AGA ACG AGA AAG ACC TGC AAA TCG AGC GAG CAG CTC CTG AAG CTA 1073 Glu Arg Thr Arg Lys Thr Cys Lys Ser Ser Glu Gin Leu Leu Lys Leu 315 320 325 CTC AGT TTA TGG AGG ATC AAA AAT GGT GAC CAA GAC ACC TTG AAG GGC 1121 Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys Gly 330 ··， 335 340 CTG ATG TAT GCC CTC AAG CAC TTG AAA ACA TCC CAC TTT CCC AAA ACT 1169 Leu Met Tyr Ala Leu Lys His Leu Lys Thr Ser His Phe Pro Lys Thr 345 350 355 360 GTC ACC CAC AGT CTG AGG AAG ACC ATG AGG TTC CTG CAC AGC TTC ACA 1217 Val Thr His Ser Leu Arg Lys Thr Met Arg Phe Leu His Ser Phe Thr 365 370 375 ATG TAC AGA CTG TAT CAG AAG CTC TTT TTA GAA ATG ATA GGG AAT CAG 1265 Met Tyr Arg Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn Gin 380 385 390 GTT CAA TCC GTG AAA ATA AGC TGC TTA TAACTAGGAA TGGTCACTGG 1312 Val Gin1 Ser Val Lys lie Ser Cys Leu 395 400 GCTGTTTCTT CA 1324 SEQ ! ID NO:124 GTATATATAA CGTGATGAGC GTACGGGTGC GGAGACGCAC CGGAGCGCTC GCCCAGCCGC 60 CGCTCCAAGC CCCTGAGGTT TCCGGGGACC ACA ATG AAC AAG' ''TTG CTG TGC TGC 114 Met Asn Lys .Leu Leu Cys Cys 1 5 GCG丨 CTC GTG · ΓΤΤ CTG GAC ATC -TCC ATT AAG TGG ACC ACC CAG GAA ACG 162 Ala ： Leu Val 1 Leu Asp lie Ser ： Lie ： Lys Trp Thr Thr Gin Glu Thr 10 15 20 -1 S - 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1221482 ABCD 七、申請專利範圍 TTT CCT CCA AAG TAC CTT CAT TAT GAC GAA GAA ACC TCT CAT CAG CTG 210 Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gin Leu 25 30 35 TTG TGT GAC AAA TGT CCT CCT GGT ACC TAC CTA AAA CAA CAC TGT ACA 255 Leu Cys Asp Lvs Cys Pro Pro Glv Thr Tyr Leu Lys Gin His Cys Thr 40 45 50 55 GCA AAG TGG AAG ACC GTG TGC GCC CCT TGC CCT GAC CAC TAC TAC ACA 306 Ala Lys Trp Lys.Thr Val Cys Ala Pro Cys Pro Asp His Tyr Tvr Thr •60 65 70 GAC AGC-TGG CAC ACC AGT GAC GAG TGT CTA TAC TGC AGC GCC GTG TGC 354 Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys 75 80 .85 AAG GAG CTG CAG TAC GTC AAG CAG GAG TGC AAT CGC ACC CAC AAC CGC 402 Lys Glu Leu Gin Tyr Val Lys Gin Glu Cys Asn Arg Thr His Asn Arg 90 95 100 GTG TGC GAA TGC AAG GAA GGG CGC TAC CTT GAG ATA GAG TTC TGC TTG 450 Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu 105 110 115 ：AAA CAT AGG AGC TGC CCT CCT GGA TTT GGA GTG GTG CAA GCT GGA ACC 49 8 Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gin Ala Gly Thr 120 125 130 135 CCA GAG CGA AAT ACA GTT TGC AAA AGA TGT CCA GAT GGG TTC TTC TCA 54 6 Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser 1 140 145 150 AAT GAG ACG TCA TCT AAA GCA CCC TGT AGA AAA CAC ACA AAT TGC AGT 594 Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser 160 165 GTC TTT GGT CTC CTG CTA ACT CAG AAA GGA AAT GCA ACA CAC GAC AAC 6 42 Val Phe Gly Leu Leu Leu Thr Gin Lys Gly Asn Ala Thr His Asp Asn 175 180 ATA TGT TCC GGA AAC AGT GAA TCA,* ACT CAA AAA TGT 'GGA ATA GAT GTT 6 90 lie Cys Ser Gly Asn Ser Glu Ser Thr Gin Lys Cys Gly lie Asp Val 185 190 195 1221482 A8 B8 C8 D8 六、申請專利範園 ACC CTG TGT GAG GAG GCA TTC TTC AGG TTT GCT GTT CCT ACA AAG TTT Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe 200 205 210 215 ACG CCT AAC TGG CTT AGT GTC TTG GTA GAC AAT TTG CCT GGC ACC AAA Thr Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys 220 225 230 GTA AAC GCA GAG AGT GTA GAG AGG ATA AAA CGG CAA CAC AGC TCA CAA Val Asn Ala Glu -Ser Val Glu Arg lie Lys Arg Gin His Ser Ser Gin 235 240 245 GAA CAG ACT TTC CAG CTG CTG AAG TTA TGG AAA CAT CAA AAC AAA GCC Glu Gin Thr Phe Gin. Leu Leu Lys Leu Trp Lys His Gin Asn Lys Ala 250. 255 260 CAA GAT ATA GTC AAG AAG ATC ATC CAA GAT ATT GAC CTC TGT GAA AAC Gin Asp lie* Val Lys Lys lie 工le Gin Asp lie Asp Leu Cys Glu Asn 265 270 275 AGC GTG CAG CGG CAC ATT GGA CAT GCT AAC CTC ACC TTC GAG CAG CTT Ser Val Gin Arg His 工le Gly His Ala Asn Leu Thr Phe Glu Gin Leu 280 285 290 295 > CGT AGC TTG ATG GAA AGC TTA CCG GGA AAG AAA GTG GGA GCA GAA GAC Arg Ser Leu.Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp 300 305 310 ATT GAA AAA ACA ATA AAG GCA TGC AAA CCC AGT GAC CAG ATC CTG AAG lie Glu Lys Thr lie Lys Ala Cys Lys Pro Ser Asp Gin lie Leu Lys ( 315 320 325 CTG CTC AGT TTG TGG CGA ATA AAA AAT GGC · GAC CAA GAC ACC TTG AAG Leu Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys 330 335 340 GGC CTA ATG CAC GCA CTA AAG CAC TCA AAG ACG TAC CAC TTT CCC AAA Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys 345 350 355 ACT GTC ACT CAG AGT CTA AAG AAG ACC ATC AGG TTC CTT CAC AGC TTC Thr Val Thr Gin Ser Leu Lys Lys Thr lie Arg Phe Leu His Ser Phe 360 365 370 _375 ACA ATG TAC AAA.TTG TAT CAG AAG TTA TTT TTA GAA ATG ATA GGT AAC Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn 380 385 390 738 7 8 6, 834 882 930 978 1026 1074 1122 11*70 1218 12661221482 No. 086104638 Patent Application B8 Chinese Patent Application Replacement (June 1993) S Announcement VI. Patent Application Fanyuan / ^ 1 Amendment Supplement 1. An isolated nucleic acid whose code has at least inhibited bone weight Absorptive peptide of osteotrophic peptide (〇PG), wherein the nucleic acid is selected from the group consisting of: a) SEQ ID NO: 120, SEQ ID NO: 122, and SEQ ID NO: 124 Nucleic acid or its complementary strand; b) stringent conditions of 5X SSC, 50% formamidine and 0.5o / 〇SDS under 4 2Ό and SEQ ID NO: 120, SEQ ID NO: 122 and SEQ ID NO: 124 Nucleic acid of the complementary strand of the polypeptide coding region shown 'that can remain hybridized under continuous washing conditions of 2X SSC at room temperature, IX SSC at 55 ° C and 0.5 × SSC at 55 ° C, SEQ ID NO: 120: ATCAAAGGCA GGGCATACTT CCTGTTGCCC AGACCTTATA TAAAACGTCA TGTTCGCCTG 60 GGCAGCAGAG AAGCACCTAG CACTGGCCCA GCGGCTGCCG CCTGAGGTTT CCAGAGGACC 120 ACA ATG AAC .AA ^ TGG CTu Tc Tu TC TTG GCA Tu Tc TGA Leu Asp lie 1 5 10 15 ATT GAA TGG ACA ACC CAG GAA ACC TTT CCT CCA AAA TAC TTG CAT TAT 216 lie Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lvs Tyr Leu His Tyr '20 25 30 GAC CCA GAA ACC GGA CGT CAG CTC TTG TGT GAC AAA TGT GCT CCT GGC 2S4 Aap Pro Glu Thr Gly Arg Gin Leu Leu Cys Asp Lya Cys Ala Pro Gly 35 40 45 ACC TAC CTA AAA * CAG CAC TGC ACA GTC AGG AGG AAG ACA CTG TGT GTC 312 Thr Tyr Leu Lvs Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val 50 55 60 CCT TGC CCT GAC TAC TCT TAT ACA GAC AGC TGG CAC ACG AGT GAT GAA 360 Pro Cys Pro Aap Tyr Ser Tyr Thr Aap Ser Trp Hia Thr Ser A3p Glu S5 70 75 O: \ 45 \ 45893-93062 l.DOC \ su This paper size is applicable to Chinese National Standard (CNS) A4 size (210 X 297 mm) 1221482 C8 D8 VI. Patent application park TGC GTG TAC TGC AGC ccc GTG TGC AAG GAA CTG CAG ACC GTG ΛΑΑ CAG 408 Cys Val Tyr Cys Ser Pro Val Cys Lys. Glu Leu Gin Thr Val Lys Gin 80 85 90 95 GAG TGC AAC CGC ACC CAC AAC CGA GTG TGC GAA TGT GAG GAA GGG CGC 456 Glu Cys Asn Arg Thr His Asn Arg Val Cvs Glu Cys Glu Glu Gly Arg 100 105 1Ϊ0 TAC CTG GAG CTC GAA TTC TGC TTG AAG CAC CGG AGC TGT CCC GCA GGC 504 Tyr Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly 115 120 125 TTG GGT GTG CTG. -CAG GCT GGG ACC CCA GAG CGA AAC ACG GTT TGC AAA 552 Leu Gly Val Leu Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys T30 135 140 AGA TGT CCG GAT GGG TTC TTC TCA GGT GAG ACG TCA TCG AAA GCA CCC 600 Arg Cys Pro Asp'Gly Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro 145 150 155 TGT AGG AAA CAC ACC AAC TGC AGC TCA CTT GGC CTC CTG CTA ATT CAG 64¾ Cys Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu lie Gin 160 165 170 175 AAA GGA GCA ACA CAT GAC AAT GTA TGT TCC GGA AAC AGA GAA GCA · 696 Lys Gly Asn Ala Thr His Asp Asn Val Cys Ser Gly Asn Arg Glu Ala 180 185 190 ACT CAA AAT TGT GGA ATA GAT GTC ACC CTG TGC GAA GAG GCA TTC TTC 744 Thr Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe * '195 200 205 AGG TTT GCT GTG CCT ACC AAG ATT ATA CCG AAT TGG CTG AGT GTT CTG 792 Arg Phe Ala Val Pro Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu 210 215 220 GTG GAC AGT TTG CCT GGG ACC AAA GTG AAT GCA GAG AGT GTA GAG AGG 840 Val Asp Ser Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg 225 230 235 ATA AAA CGG AGA CAC AGC TCG CAA GAG CAA ACT TTC CAG CTA CTT AAG 888 lie Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys 240 245 250 255 CTG TGG AAG CAT CAA AAC AGA GAC CAG GAA • ATG GTG AAG AAG ATC ATC 936 Leu Trp Lys His Gin Asn Arg Asp Gin Glu Met Val Lys Lys lie lie 260 265 270 CAA GAC ATT GAC CTC TGT GAA AGC AGT GTG CAA CGG CAT ATC GGC CAC 984 Gin Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His lie Gly His 275 280 285 • 2- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1221482 A8 B8 C8 D8 々, patent application GGC AAC CTC ACC ACA GAG CAG CTC CGC ATC TTG ATG GAG AGC TTG CCT Ala Asn Leu Thr Thr Glu Gin Leu Arg'Ile Leu Met Glu Ser Leu Pro 290 295 300 GGG AAG AAG ATC AGC CCA GAC GAG ATT GAG AGA ACG AGA AAG ACC TGC Giy Lys Lys lie Ser Pro Asp Glu lie Glu Arg Thr Arg Lys Thr Cys 305 310 315 AAA CCC AGC GAG CAG CTC CTG AAG CTA CTG AGC TTG TGG AGG ATC AAA Lys Pro Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys 320 325 330 335 AAT GGA GAC CAA * GAC ACC TTG AAG GGC CTG ATG TAC GCA CTC AAG CAC Asn Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His 340 345 350 * i- TTG AAA GCA -TAC .CAC TTT CCC AAA ACC GTC ACC CAC AGT CTG AGG AAG Leu Lys Ala Tyr His Phe Pro Lys Thr Val Thr His Ser Leu Arg Lys 355 360 365 ACC ATC AGG TTC TTG CAC AGC TTC ACC ATG TAC CGA TTG TAT CAG AAA Thr lie Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys 370 375 380 CTC TTT CTA GAA ATG ATA GGG AAT CAG GTT CAA TCA GTG AAG ATA AGC Leu Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser 385 390 395 TGC TTA TAGTTAGGAA TGGTCACTGG GCTGTTTCTT CAGGATGGGC CAACACTGAT Cys Leu 400 GGAGCAGATG GCTGCTTCTC CGGCTCTTGA AATGGCAGTT GATTCCTTTC TCATCAGTTG GTGGGAATGA AGATCCTCCA GCCCAACACA CACACTGGGG AGTCTGAGTC AGGAGAGTGA GGCAGGCTAT TTGATAATTG TGCAAAGCTG CCAGGTGTAC ACCTAGAAAG TCAAGCACCC TGAGAAAGAG GATATTTTTA TAACCTCAAA CATAGGCCCT TTCCTTCCTC TCCTTATGGA TGAGTACTCA GAAGGCTTCT ACTATCTTCT GTGTCATCCC TAGATGAAGG CCTCTTTTAT TTATTTTTTT ATTCTTTTTT TCGGAGCTGG GGACCGAACC CAGGGCCTTG CGCTTGCGAG GCAAGTGCTC TACCACTGAG CTAAATCTCC AACCCCTGAA GGCCTCTTTC TTTCTGCCTC TGATAGTCTA TGACATTCTT TTTTCTACAA TTCGTATCAG GTGCACGAGC CTTATCCCAT TTGTAGGTTT CTAGGCAAGT TGACCGTTAG CTATTTTTCC CTCTGAAGAT TTGATTCGAG TTGCAGACTT GGCTAGACAA GCAGGGGTAG GTTATGGTAG TTTATTTAAC AGACTGCCAC -3- this paper scales applicable Chinese national standard (CNS) A4 size (210X 297 mm) 1,032,108,011,281,176 1224 lil2 1320 1376 1436 1496 1556 1616 1676 1736 1796 1856 1916 1976 1221482 A8 B8 C8 D8 VI. Application scope CAGGAGTCCA GTGTTTCTTG TTCCT CTGTA GTTGTACCTA AGCTGACTCC AAGTACATTT 2036 AGTATGAAAA ATAATCAACA AATTTTATTC CTTCTATCAA CATTGGCTAG CTTTGTTTCA 2096 GGGCACTAAA AGAAACTACT ATATGGAGAA AGAATTGATA TTGCCCCCAA CGTTCAACAA 2156 CCCAATAGTT TATCCAGCTG TCATGCCTGG TTCAGTGTCT ACTGACTATG CGCCCTCTTA 2216 TTACTGCATG CAGTAATTCA ACTGGAAATA GTAATAATAA TAATAGAAAT AAAATCTAGA 2276 CTCCATTGGA TCTC.TCTGAA TATGGGAATA TCTAACTTAA GAAGCTTTGA GATTTCAGTT 2336 GTGTTAAAGG CTTTTATTAA AAAGCTGATG CTCTTCTGTA AAAGTTACTA ATATATCTGT 2396 AAGACTATTA CAGTATTGCT ATTTATATCC ATCCAG 2432 SEQ ID NO: 122 CCTTATATAA ACGTCATGAT TGCCTGGGCT GCAGAGACGC ACCTAGCACT GACCCAGCGG *. 60 CTGCCTCCTG AGGTTTCCCG AGGACCACA ATG AAC AAG Met Asn Lys 1 TGG CTG CTC TGC Trp Le CAC GAC TAC Lec Cyc ATC TGG ACA ACC CAG GAA ACC CTT 161 Leu Leu Val Leu Leu Asp lie lie Glu Trp Thr Thr Gin Glu Thr Leu 10 15 20 CCT CCA AAG TAC TTG CAT TAT GAC CCA GAA ACT GGT CAT CAG CTC CTG 209 Pro Pro Lys Tyr Leu His Tyr Asp Pro Glu Thr Gly His Gin Le u Leu 25 30 35 40 TGT GAC AAA TGT GCT CCT GGC ACC TAC CTA AAA CAG CAC TGC ACA GTG 257 Cys Asp Lys Cys Ala Pro Gly Thr Tyr Leu Lys Gin Kis Cys Thr Val 45 50 55 AGG AGG AAG ACA TTG TGT GTC CCT TGC CCT GAC CAC TCT TAT ACG GAC 305 Arg Arg Lys Thr Leu Cys Val Pro Cys Pro Asp His Ser Tyr Thr Asp βα 65 * * '70 AGC TGG CAC ACC AGT GAT GAG TGT GTG TAT TGC AGC CCA GTG TGC AAG 353 Ser Trp His Thr Ser Asp Glu Cys Val Tyr Cys Ser Pro Val Cys Lys 75 80 85 -4- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A8 B8 C8 D8 VI. Patent application scope GAA CTG CAG TCC GTG AAG CAG GAG TGC AAC CGC ACC CAC AAC CGA GTG Glu Leu Gin Ser Val Lys Gin Glu Cys Asn Arg Thr His Asn Arg Val 90 95 100 TGT GAG TGT GAG GAA GGG CGT TAC CTG GAG ATC GAA TTC TGC TTG AAG Cys Glu Cys Glu Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu Lys 105 110 115 120 CAC CGG AGC TGT CCC CCG GGC TCC GGC GTG GTG CAA GCT GGA ACC CCA His Arg Ser Cys Pro Pro Gly Ser Gly Val V al Gin Ala Gly Thr Pro 125 130 135 GAG CGA · AAC ACA GTT TGC AAA AAA TGT CCA GAT GGG TTC TTC TCA GGT Glu Arg Asn " Thr-Val Cys Lys Lys Cys Pro Asp Gly Phe Phe Ser Gly 140 145 150 GAG ACT TCA TCG AAA GCA CCC TGT ATA AAA CAC ACG AAC TGC AGC ACA Glu Thr Ser Ser Lys Ala Pro Cys lie Lys His Thr Asn Cys Ser Thr 155 160 165 TTT GGC CTC CTG CTA ATT CAG AAA GGA AAT GCA ACA CAT GAC AAC GTG Phe Gly Leu Leu Leu lie Gin Lys Gly Asn Ala Thr His Asp Asn Val 17 0 175 180 TGT TCC GGA 'AAC AGA GAA GCC ACG CAA AAG TGT GGA ATA GAT GTC ACC Cys Ser Gly Asn Arg Glu Ala Thr Gin Lys Cys Gly lie Asp Val Thr 185 190 195 200 CTG TGT GAA GAG GCC TTC TTC AGG TTT GCT GTT CCT ACC AAG ATT ATA Leu Cys Glu Glu Ala Phe Phe Arg Phe • Ala Val Pro Thr Lys lie lie 205 210 215 CCA AAT TGG CTG AGT GTT TTG GTG GAC AGT TTG CCT GGG ACC AAA GTG Pro Asn Trp Leu Ser Val Leu Val Asp Ser Leu Pro Gly Thr Lys Val 220 225 230 AAT GCC GAG AGT GTA GAG AGG ATA AAA CGG AGA CAC AGC TCA CAA GAG Asn Ala Glu Ser Val Glu Arg lie Lys Arg Arg His Ser Ser Gin Glu 235 240 245 CAA ACC TTC CAG CTG CTG AAG CTG TGG AAA CAT CAA; AAC AGA GAC .CAG Gin Thr Phe Gin Leu Leu Lys Leu, Trp Lys His Gin Asn Arg Asp Gin 250 255 260 GAA ATG GTG AAG AAG ATC ATC CAA GAC ATT GAC CTC TGT GAA AGC AGC Glu Met Val Lys Lys lie lie Gin Asp lie Asp Leu Cys Glu Ser Ser 265 27 0 275 280 401 449 497 545 593 641 689 73 * 7 785 833 881 929 -5- This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm) 1221482 as Jd8 C8 D8 Six The scope of patent application GTG CAG CGG CAT CTC GGC CAC TCG AAC CTC ACC ACA GAG CAG CTT CTT 977 Vai Gin Arg His Leu Gly His Ser Asn Leu Thr Thr Glu Gin Leu Leu 285 290 295 GCC TTG ATG GAG AGC CTG CCT GGG AAG AAG ATC AGC CCA GAA GAG ATT 1025 Ala Leu Met Glu Ser Leu Pro Gly Lys Lys lie Ser Pro Glu Glu lie 300 305 310 GAG AGA ACG AGA AAG ACC TGC AAA TCG AGC GAG CAG CTC CTG AAG CTA 1073 Glu Arg Thr Arg Lys Thr Cys Lys Ser Ser Glu Gin Leu Leu Lys Leu 315 320 325 CTC AGT TTA TGG AGG ATC AAA AAT GGT GAC CAA GAC ACC TTG AAG GGC 1121 Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys Gly 330 ..., 335 340 CTG ATG TAT GCC CTC AAG CAC TTG AAA ACA TCC CAC TTT CCC AAA ACT 1169 Leu Met Tyr Ala Leu Lys His Leu Lys Thr Ser His Phe Pro Lys Thr 345 350 355 360 GTC ACC CAC AGT CTG AGG AAG ACC ATG AGG TTC CTG CAC AGC TTC ACA 1217 Val Thr His Ser Leu Arg Lys Thr Met Arg Phe Leu His Ser Phe Thr 365 370 375 ATG TAC AGA CTG TAT CAG AAG CTC TTT TTA GAA ATG ATA GGG AAT CAG 1265 Met Tyr Arg Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn Gin 380 385 390 GTT CAA TCC GTG AAA ATA AGC TGC TTA TAACTAGGAA ValGGinACT1 13 Lys lie Ser Cys Leu 395 400 GCTGTTTCTT CA 1324 SEQ! ID NO: 124 GTATATATAA CGTGATGAGC GTACGGGTGC GGAGACGCAC CGGAGCGCTC GCCCAGCCGC 60 CGCTCCAAGC CCCTGAGGTT TCCGGGGACC ACA ATG AAC AAG '' 'TTG CTG TGC TGC 114 Met Asn Lys .Leu Leu Cys Cys 1 5GCG 丨 CTCT ACC ACC CAG GAA ACG 162 Ala: Leu Val 1 Leu Asplie Ser: Lie: Lys Trp Thr Thr Gin Glu Thr 10 15 20 -1 S-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) ) 1221482 ABCD VII. Patent application scope TTT CCT CCA AAG TAC CTT CAT TAT GAC GAA GAA ACC TCT CAT CAG CTG 210 Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gin Leu 25 30 35 TTG TGT GAC AAA TGT CCT CCT GGT ACC TAC CTA AAA CAA CAC TGT ACA 255 Leu Cys Asp Lvs Cys Pro Pro Glv Thr Tyr Leu Lys Gin His Cys Thr 40 45 50 55 GCA AAG TGG AAG ACC GTG TGC GCC CCT TGC CCT GAC CAC TAC TAC ACA 306 Ala Lys Trp Lys.Thr Val Cys Ala Pro Cys Pro Asp His Tyr Tvr Thr • 60 65 70 GAC AGC-TGG CAC ACC AGT GAC GAG TGT CTA TAC TGC AGC GCC GTG TGC 354 Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys 75 80 .85 AAG GAG CTG CAG TAC GTC AAG CAG GAG TGC AAT CGC ACC CAC AAC CGC 402 Lys Glu Leu Gin Tyr Val Lys Gin Glu Cys Asn Arg Thr His Asn Arg 90 95 100 GTG TGC GAA TGC AAG GAA GGG CGC TAC CTT GAG ATA GAG TTC TGC TTG 450 Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu 105 110 115: AAA CAT AGG AGC TGC CCT CCT GGA TTT GGA GTG GTG CAA GCT GGA ACC 49 8 Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gin Ala Gly Thr 120 125 130 135 CCA GAG CGA AAT ACA GTT TGC AAA AGA TGT CCA GAT GGG TTC TTC TCA 54 6 Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser 1 140 145 150 AAT GAG ACG TCA TCT AAA GCA CCC TGT AGA AAA CAC ACA AAT TGC AGT 594 Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser 160 165 GTC TTT GGT CTC CTG CTA ACT CAG AAA GGA AAT GCA ACA CAC GAC AAC 6 42 Val Phe Gly Leu Leu Leu Thr Gin Lys Gly Asn Ala Thr His Asp Asn 175 180 AT A TGT TCC GGA AAC AGT GAA TCA, * ACT CAA AAA TGT 'GGA ATA GAT GTT 6 90 lie Cys Ser Gly Asn Ser Glu Ser Thr Gin Lys Cys Gly lie Asp Val 185 190 195 1221482 A8 B8 C8 D8 ACC CTG TGT GAG GAG GCA TTC TTC AGG TTT GCT GTT CCT ACA AAG TTT Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe 200 205 210 215 ACG CCT AAC TGG CTT AGT GTC TTG GTA GAC AAT TTG CCT GGC ACC AAA Thr Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys 220 225 230 GTA AAC GCA GACA GAG AGT GTA GAG AGG ATA AAA CGG CAA CAC AGC TCA CAA Val Asn Ala Glu -Ser Val Glu Arg lie Lys Arg Gin His Ser Ser Gin 235 240 245 GAA CAG ACT TTC CAG CTG CTG AAG TTA TGG AAA CAT CAA AAC AAA GCC Glu Gin Thr Phe Gin. Leu Leu Lys Leu Trp Lys His Gin Asn Lys Ala 250. 255 260 CAA GAT ATA GTC AAG AAG ATC ATC CAA GAT ATT GAC CTC TGT GAA AAC Gin Asp lie * Val Lys Lys lie gin Asp lie Asp Leu Cys Glu Asn 265 270 275 AGC GTG CAG CGG CAC ATT GGA CAT GCT AAC CTC ACC TTC GAG CAG CTT Ser Val Gin Arg His Gly His Ala Asn Leu Thr Phe Glu Gin Leu 280 285 290 295 > CGT AGC TTG ATG GAA AGC TTA CCG GGA AAG AAA GTG GGA GCA GAA GAC Arg Ser Leu.Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp 300 305 310 ATT GAA AAA ACA ATA AAG GCA TGC AAA CCC AGT GAC CAG ATC CTG AAG lie Glu Lys Thr lie Lys Ala Cys Lys Pro Ser Asp Gin lie Leu Lys (315 320 325 CTG CTC AGT TTG TGG CGA ATA AAA AAT GGCGAC CAA GAC ACC TTG AAG Leu Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys 330 335 340 GGC CTA ATG CAC GCA CTA AAG CAC TCA AAG ACG TAC CAC TTT CCC AAA Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys 345 350 355 ACT GTC ACT CAG AGT CTA AAG AAG ACC ATC AGG TTC CTT CAC AGC TTC Thr Val Thr Gin Ser Leu Lys Lys Thr lie Arg Phe Leu His Ser Phe 360 365 370 _375 ACA ATG TAC AAA.TTG TAT CAG AAG TTA TTT TTA GAA ATG ATA GGT AAC Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn 380 385 390 738 7 8 6, 834 882 930 978 1026 1074 1122 11 * 70 1218 1266

裝 iiOutfit ii

-8- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1221482 A8 B8 C8 D8 六、申請專利範園 CAG GTC CAA TCA GTA AAA ATA AGC TGC TTA TAACTGGAAA TGGCCATTGA 1316 Gin Val Gin Ser Val Lys lie Ser Cys Leu 395 400 GCTGTTTCCT CACAATTGGC GAGATCCCAT GGATGATAA 1355 C)可於42°c下5X SSC、50%甲醯胺及0.5% SDS之嚴格條件與編碼如下示自胺基酸148至33 7(含）之核苷酸雜合，並可於室溫下2X SSC、55°C下IX SSC及55 它下0.5X SSC之連續沖洗條件下保持雜合狀態之核酸， 148 178 208 238 268 298 FRI -1 XLLVFLDIIEWTTQETFPPKYLHYDPETGRQLLCDKCAPGTYLKQHCTVRRKTLCVPCPD I ： I ll：ll I I I 1：1 ： I I HALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCED 30 40 50 60 70 80 328 YSYTDSVmTS .:11:1: STYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPL 90 100 110 120 130 140 :及 d) (a)、（b)及（c)之核酸之簡併核酸。 2 ·根據申請專利範圍第1項之核酸，其為cDNA，基因體 DNA，合成DNA或RNA。 3 · 一種為根據申請專利範圍第1項所述核酸，所編碼的多肽。 -9- 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) 1221482 as B8 C8 __D8 六、申請專利範圍 4 .根據申請專利範圍第i項之核酸，其包括一或多種適於大腸桿菌(Escherichia coH)表現之密碼子。 5.根據申請專利範圍第1項之核酸，其具有接著在其上的一種可偵檢之標記。 6 ·根據申請專利範圍第1項之核酸，其包括SEq id NO : 120，SEQ ID NO : 122 或 SEQ ID NO : 124 的多肽編碼區。 7 ·根據申請專利範圍第6項之核酸，其具有SEQ ID NO : 124所示從核苷酸158-1297之序列。 8 · —種表現載體，其包括根據申請專利範圍第1項之核酸。 9 ·根據申請專利範圍第8項之表現載體，其中該核酸包括 SEQ ID NO : 124中所示之多肽編碼區。 10. —種經根據申請專利範圍第8項所述表現載體轉形或轉染過之宿主細胞。 11·根據申請專利範圍第1 〇項之宿主細胞，其為一種真核生物細胞。 12·根據申請專利範圍第1 1項之宿主細胞，其選自ch〇， COS，293，3T3，CV-1和BHK細胞所成組合之中者。 13·根據申請專利範圍第1 〇項之宿主細胞，其為一種原核生物細胞。 14.根據申請專利範圍第1 3項之宿主細胞，其為大腸桿菌。 15· —種產生OPG的方法，其包括：在適當的營養條件生長經用根據申請專利範圍第1項所 -10--8- This paper size applies to Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 1221482 A8 B8 C8 D8 VI. Patent Application Fan Garden CAG GTC CAA TCA GTA AAA ATA AGC TGC TTA TAACTGGAAA TGGCCATTGA 1316 Gin Val Gin Ser Val Lys lie Ser Cys Leu 395 400 GCTGTTTCCT CACAATTGGC GAGATCCCAT GGATGATAA 1355 C) Strict conditions and codes for 5X SSC, 50% formamidine, and 0.5% SDS at 42 ° C are shown below from amino acids 148 to 33 7 (including Nucleotides that are heterozygous and can remain hybridized under continuous washing conditions of 2X SSC at room temperature, IX SSC at 55 ° C and 55X SSC at 55 ° C, 148 178 208 238 268 298 FRI- 1 XLLVFLDIIEWTTQETFPPKYLHYDPETGRQLLCDKCAPGTYLKQHCTVRRKTLCVPCPD I: I ll: ll III 1: 1: II HALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCED 30 40 50 60 70 80 328 YSYTDSVmTS:. 11: 1: STYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPL 90 100 110 120 130 140: and d) (a), (b) and (c ) Of a degenerate nucleic acid. 2. The nucleic acid according to item 1 of the scope of patent application, which is cDNA, genomic DNA, synthetic DNA or RNA. 3. A polypeptide encoded by the nucleic acid described in item 1 of the patent application. -9- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 as B8 C8 __D8 VI. Application for patent scope 4. According to the nucleic acid of item i of the patent scope, it includes one or more suitable for large intestine Codons expressed by Escherichia coH. 5. The nucleic acid according to item 1 of the scope of patent application, which has a detectable mark next to it. 6. The nucleic acid according to item 1 of the scope of patent application, which comprises a polypeptide coding region of SEq id NO: 120, SEQ ID NO: 122 or SEQ ID NO: 124. 7. The nucleic acid according to item 6 of the scope of patent application, which has the sequence shown in SEQ ID NO: 124 from nucleotides 158-1297. 8-A performance vector comprising a nucleic acid according to item 1 of the scope of patent application. 9. The expression vector according to item 8 of the application, wherein the nucleic acid includes the polypeptide coding region shown in SEQ ID NO: 124. 10.-A host cell transformed or transfected with the expression vector according to item 8 of the scope of the patent application. 11. The host cell according to item 10 of the application, which is a eukaryotic biological cell. 12. The host cell according to item 11 of the scope of patent application, which is selected from the group consisting of ch0, COS, 293, 3T3, CV-1 and BHK cells. 13. A host cell according to item 10 of the application, which is a prokaryotic biological cell. 14. The host cell according to item 13 of the application, which is E. coli. 15. · A method for producing OPG, comprising: growing under appropriate nutritional conditions according to item 1 of the patent application scope -10-

1221482 A8 B8 C8 D8 六、申請專利範園述核酸轉形或轉染過之宿主細胞；及分離出該核酸表現所得多肽產物。 16. —種經分離之OPG多肽，其可增加骨密度或抑制骨重吸收，具有如下所示一或多者之胺基酸序列： (a) 如 SEQ ID NO: 121、SEQ ID NO: 123 或 SEQ ID NO: 125所示胺基酸序列第1至401殘基 (含）； (b) 如 SEQ ID NO: 121、SEQ ID NO: 123 或 SEQ ID NO: 125所示胺基酸序列第22至401殘基 (含）；及 (c) 含（a)或（b)所載片段之胺基酸序列： SEQ ID NO: 121: Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Phe Leu Asp lie lie 15 10 15 Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 1 20 25 30 Pro Glu Thr Gly Arg Gin Leu Leu Cy.s Asp Lys Cys Ala Pro Gly Thr 35 40 45 Tyr Leu Lys Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val Pro 50 55 60 Cys Pro Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Thr Val Lys Gin Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Glu Glu Gly Arg Tyr 100 105 110 Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Leu 115 120 125 -11 - 本紙張尺度適用中國國家標準(CNS) A4規格(210x 297公釐) 1221482 A8 B8 C8 D8 六、申請專利範圍 Gly Val Leu Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lvs Arg 130 135 140 - Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His Thr Asn Cys Ser Ser Leu Gly J-eu Leu Leu lie Gin Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Val Cys Ser Gly Asn Arg Glu Ala Thr 180 185 190 Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro.Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu Val .210 215 220 Asp Ser Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240 Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255 Trp Lys, His Gin Asn Arg Asp Gin'Glu Met Val Lys Lys lie lie Gin 260 265 270 Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His lie Gly His Ala 275 280 285 Asn Leu Thr Thr Glu Gin Leu Arg lie Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys lie Ser Pro Asp Glu lie Glu Arg Thr Arg Lys Thr Cys Lya 305 310 315 320 Pro Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335 Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu 340 345 350 Lys Ala Tyr His Phe Pro Ly^ Thr Val Thr His Ser Leu Arg Lys Thr 355 360 365 工le Arg Phe. Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu 370 375 380 · Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys 385 390 395 400 Leu -12- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1221482 A8 B8 C8 D8 六、申請專利範圍 SEQ ID NO: 123: Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Leu Leu Asp lie Ide 1 5 10 15 Glu Trp Thr Thr Gin Glu Thr Leu Pro Pro Lys Tyr Leu His Tyr Asp . 20 25 30 Pro Glu Thr Gly His Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly Thr 35 40 45 Tyr Leu Lys Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val P.ro 50 55 60 Cys Pro- As-p. His Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Ser Val Lys Gin Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Glu Glu Gly Arg Tyr 100 105 no Leu Glu lie Glu Phe Cys.Leu Lys His Arg Ser Cys Pro Pro Gly Ser 115 120 125 Gly Val. Val Gin Ala Gly Thr Pro Glu 'Arg Asn Thr Val Cys Lys Lys 130 135 140 i Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 lie Lys His Thr Asn Cys Ser Thr Phe Gly Leu Leu Leu lie Gin Lys 165 170 175 Gly Asn Ala Thr His Asp Asn ^al Cys Ser Gly Asn Arg Glu Ala Thr 180 185 190 Gin Lys Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu Val 210 - 215 220 Asp Ser Leu Pro Glv Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240 Lya Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255 -13- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐)1221482 A8 B8 C8 D8 VI. Patent application Fanyuan describes the nucleic acid transformed or transfected host cells; and isolates the polypeptide product obtained by the expression of the nucleic acid. 16. An isolated OPG polypeptide, which can increase bone density or inhibit bone resorption, has one or more of the amino acid sequences shown below: (a) As SEQ ID NO: 121, SEQ ID NO: 123 Or residues 1 to 401 of the amino acid sequence shown in SEQ ID NO: 125 (inclusive); (b) as shown in SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 125 Residues 22 to 401 (inclusive); and (c) amino acid sequences containing the fragments contained in (a) or (b): SEQ ID NO: 121: Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Phe Leu Asp lie lie 15 10 15 Glu Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 1 20 25 30 Pro Glu Thr Gly Arg Gin Leu Leu Cy.s Asp Lys Cys Ala Pro Gly Thr 35 40 45 Tyr Leu Lys Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val Pro 50 55 60 Cys Pro Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Thr Val Lys Gin Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Glu Glu Gly Arg Tyr 100 105 110 Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Leu 115 120 125 -11-This paper size applies to Chinese National Standard (CNS) A4 size (210x 297 mm) 1221482 A8 B8 C8 D8 VI. Patent application scope Gly Val Leu Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lvs Arg 130 135 140-Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His Thr Asn Cys Ser Ser Leu Gly J-eu Leu Leu lie Gin Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Val Cys Ser Gly Asn Arg Glu Ala Thr 180 185 190 Gin Asn Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro. Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu Val .210 215 220 Asp Ser Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240 Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255 Trp Lys , His Gin Asn Arg Asp Gin'Glu Met Val Lys Lys lie lie Gin 260 265 270 Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His lie Gly His Ala 275 280 285 Asn Leu Thr Thr Glu Gin Leu Arg lie Leu Met Glu Ser Le u Pro Gly 290 295 300 Lys Lys lie Ser Pro Asp Glu lie Glu Arg Thr Arg Lys Thr Cys Lya 305 310 315 320 Pro Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335 Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu 340 345 350 Lys Ala Tyr His Phe Pro Ly ^ Thr Val Thr His Ser Leu Arg Lys Thr 355 360 365 industrial Arg Phe. Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu 370 375 380Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys 385 390 395 400 Leu -12- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1221482 A8 B8 C8 D8 VI. Application scope of SEQ ID NO: 123: Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Leu Leu Asp lie Ide 1 5 10 15 Glu Trp Thr Thr Gin Glu Thr Leu Pro Pro Lys Tyr Leu His Tyr Asp. 20 25 30 Pro Glu Thr Gly His Gin Leu Leu Cys Asp Lys Cys Ala Pro Gly Thr 35 40 45 Tyr Leu Lys Gin His Cys Thr Val Arg Arg Lys Thr Leu Cys Val P.ro 50 55 60 Cys Pro- As- p. His Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Val Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Ser Val Lys Gin Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Glu Glu Gly Arg Tyr 100 105 no Leu Glu lie Glu Phe Cys.Leu Lys His Arg Ser Cys Pro Pro Gly Ser 115 120 125 Gly Val. Val Gin Ala Gly Thr Pro Glu 'Arg Asn Thr Val Cys Lys Lys 130 135 140 i Cys Pro Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 lie Lys His Thr Asn Cys Ser Thr Phe Gly Leu Leu Leu lie Gin Lys 165 170 175 Gly Asn Ala Thr His Asp Asn ^ al Cys Ser Gly Asn Arg Glu Ala Thr 180 185 190 Gin Lys Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro Thr Lys lie lie Pro Asn Trp Leu Ser Val Leu Val 210-215 220 Asp Ser Leu Pro Glv Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240 Lya Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255 -13- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

裝訂Binding

六、申請專利範園 Trp Lys His Gin Asn Arg Asp Gin Glu Met Val Lys Lys lie lie Gin 260 265 270 Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His Leu Gly His Ser 275 280 285 Asn Leii Thr Thr Glu Gin Leu Leu Ala Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys lie Ser Pro Glu Glu lie Glu Arg Thr Arg Lys Thr Cys Lys 305 310 315 320 Ser Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335 Gly Asp Gin Asp Thr Leu 'Lys Gly Leu Met Tyr Ala Leu Lys His Leu 340 345 350 Lys Thr Ser His Phe Pro Lys Thr Val Thr His Ser.Leu Arg Lys Thr 355 360 365 Met Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu 370 375 380 Phe Leu Glu Met 工le Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys 385 ' 390 395 400 Leu SEQBDNO:125: Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie 15 10 15 Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr L'eu His Tyr Asp * 20 25 30 Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 - 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu ** 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 -14- 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) 1221482 A8 B8 C8 D8 、申請專利範圍 Leu Glu lie Glu Phe Cya Leu Lys His Arg Ser Cya Pro Pro Gly Phe 115 120 125 31y Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 ** 150 155 160 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys 165 170 175 Gly Asn Ala- Thr His Asp Asn lie Cys Ser Gly Asn Ser Glu Ser Thr ...180 185 190 Gin Lvs Cvs Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 一 195 * 200 205 Phe Ala Val Pro Thr'Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 ^ 215 220 Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240 Lys Arg Gin His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255 Trp Lys His Gin Asn Lys Ala Gin Asp lie Val Ly.s Lys lie lie Gin 260 265 270 Asp lie Asp Leu Cys Glu Asn Ser Val Gin Arg His lie Gly Kis Ala 275 280 285 Asn Leu Thr Phe Glu Gin Leu Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys Val Gly Ala Glu Asp lie Glu Lys Thr lie Lys Ala Cys Lys 305 310 315 320 Pro Ser Asp Gin lie Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335 Gly Asp Gin Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser 340 345 350 Lys Thr Tvr His Phe Pro Lys Thr Val Thr Gin -.Ser Leu Lys .Lys Thr 355. 360 365 lie Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leil 370 375 380 Phe Leu Glu Met He Gly Asn Gin Val Gin Ser Val Lys lie Ser Cya 385 390 395 400 O Leu -15- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) D8 六、申請專利範圍 17·根據申請專利範圍第16項之多肽，其為哺乳動物OPG。 18·根據申請專利範圍第1 7項之多肽，其為人類〇PG。 19·根據申請專利範圍第1 6項之多肽，其實質地不含其他人類蛋白質。 2〇·根據申請專利範圍第1 6項之多肽，其具有SEQ ID NO : 125中所示殘基22_4〇1(包括殘基22和4〇1)之胺基酸序列。 21.根據申請專利範圍第1 6項之多肽，其具有SEq id NO : 125中所示殘基32-401(包括殘基32和401)之胺基酸序列。 22·根據申請專利範圍第1 6項之多肽，其特徵在於其為外源 DNA序列的表現產物。 23·根據申請專利範圍第2 2項之多肽，其中該DNA為cDNA，基因體DNA或合成DNA。 24·根據申請專利範圍第1 6項之多肽，其更經水溶性聚合物改質過。 25·根據申請專利範圍第2 4項之多肽，其中該水溶性聚合物為聚乙二醇。 26. —種多肽，其包含：一有至少約164胺基酸的胺基酸序列，其中包括四個具有腫瘤壞死因子受體細胞外區所含富含半胱胺酸域的特性之富含半胱胺酸的區域，其中該胺基酸序列為S e Q ID NO: 121、SEQ ID NO: 123 及 SEQ ID NO: 125 所示胺基酸序列之一部份或全部；及 -16- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 六、申請專利範園一增加骨密度之活性。 27. —種多肽，其具有 SEQ ID NO : 121，SEQ ID NO : 123或 SEQ ID NO : 125中所示胺基酸序列，其具有在殘基22處之胺基端，且其中1至216胺基酸從羧基端缺失掉。 28·根據申請專利範圍第27項之多肽，其包括殘基22_185(包括殘基22和185)，22-189(包括22和189)，22-194(包含殘基22和194)，或22-201(包含殘基22和201)諸胺基酸序列。 29·根據申請專利範圍第2 8項之多肽，其更包括從羧基端延伸出的人類IgGl所含Fc區。 30· — 種多肽，其具有SEQ ID NO : 121，SEQ ID NO : 123 或 SEQ ID NO : 125中所示胺基酸序列且在殘基22處具有一胺基端，其中從胺基端缺失掉1至10胺基酸。 31. 根據申請專利範圍第3 0項之多肽，其另從羧基端缺失掉 1至216胺基酸。 32. 根據申請專利範圍第3 0項之多肽，其具有殘基27-1 85(包含殘基27和185)，27-189(包含殘基27和189)，27-194(包括殘基27和194)，27-401(包含殘基27和401)，或32-401(包含殘基32和401)之胺基酸序列。 33. 根據申請專利範圍第3 2項之多肽，其更包括從其叛基端延伸出的人類IgGl所含Fc區。 34. —種多肽，其係選自下列所成組合之中者： huOPG [22-201]-Fc huOPG [22-401]-Fc -17- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1221482 A8 B8 C8 D8 六、申請專利範圍 huOPG [22-180]-Fc huOPG met [22-401]-Fc huOPG Fc-met [22-201] huOPG met [22-185] huOPG met [22-189] huOPG met [22-194] huOPG met [27-185] huOPG met [27-189] huOPG met [27-194] huOPG met [32-401] huOPG met-lys [22-401] huOPG met [22-401] huOPG met [22-401 ]-Fc(P25A) huOPG met [22-401] (P25A) huOPG met [22-401] (P26A) huOPG met [22-401] (P26D) huOPG met [22-194] (P25A) huOPG met [22-194] (P26A) huOPG met met-(lys)3 [22-401] huOPG met met-arg-gly-ser-(his)6[22-401 ] j 其中huOPG含有SEQ ID NChl25所示胺基酸序歹ij。 35· —種編碼根據申請專利範圍第3 4項所述多肽之核酸。 36. —種用以評估候選物質結合到OPG的能力之方法，其包 -18- 本紙張尺度適用中國國家標準(CNS) Α4規格(210X297公釐) 1221482 A8 B8 C8 _ D8 I、申請專利範圍 — 括·· 用根據申請專利範圍第3、16、26、27、3〇或34項之 OPG與該候選物質在可促成結合的條件下培育；及測量被結合的該物質。 37· —種調節動物體内0PG含量之醫藥組合物，其含有治療有效量之根據申請專利範圍第1項之核酸。 38.根據申請專利範圍第37項之醫藥組合物，其中該核酸可促成組織OPG水平之增加。 39·根據申請專利範圍第38項之醫藥組合物，其中該動物為人類。 40· —種治療骨病之醫藥組合物，其包括治療有效量之根據申請專利範圍第3、16、26、27、30或34項之多肽與醫藥可接受的載體，佐劑，落解化劑，安定劑及/或抗氧化物。 4L根據申請專利範園第4〇項之組合物，其中該〇pG為人類 OPG。 42·根據申請專利範圍第41項之組合物，其中該〇pG具有下示之胺基酸序列々、申請專利範圍 79Q 810 830 AAAGTGAATGCCGAGAGTGTAGAGAGGATAAAACGGAGACACAGCTCACAAGAGCAAACC KVNAESVERIKRRHSSQEQT 850 870 890 TTCCAGCTGCTGAAGCTGTGGAAACATCAAAACAGAGACCAGGAAATGGTGAAGAAGATC FQ*Lr.LKLWKHQNRDQEMVKKI 910 930 950 ATCCAAGACATTGACCTCTGTGAAAGCAGCGTGCAGCGGCATCTCGGCCACTCGAACCTC IQDIDLCESSVQRHLGHSiiL 970 990 1010 ACCACAGAGCAGCTTCTTGCCTTGATGGAGAGCCTGCCTGGGAAGAAGATCAGCCCAGAA TTEQLLALHESLPGKKISPE -1030 1050 ·· 1070 GAGATTGAGAGAACGAGAAAGACCTGCAAATCGAGCGAGCAGCTCCTGAAGCTACTCAGT E I^ERTRKTCKSS EQLLKLLS^ 1090 1110 1130 TTATGGAGGATCAAAAATGGTCACCAAGACACCTTGAAGGGCCTGATGTATGCCCTCAAG LW RIKNGDQDTLKGLMYALK 1150 1170 1190 CACTTGAAAACATCCCACTTrCCCAAAACTGTCACCCACAGTCTGAGGAAGACCATGAGG HLKTSHFPKTVTHSLRKTMR 1210 1230 1250 TTCCTGCACAGCTTCACAATGTACAGACTGTATCAGAAGCTCTTTTTAGAAATGATAGGG FLH. SFTMYRLYQ KLFLEMI G 1270 1290 1310 AATCAGGTTCAATCCGTGAAAATAAGCTGCTTATAACTAGGAATGGTCACTGGGCTGTTT NQVQSVKISCL 43. —種治療骨病之醫藥組合物，其含有治療有效量的根據申請專利範圍第1 6項之多肽。 44. 根據申請專利範圍第43項之.醫藥組合物，其中該多肽為人類OPG。 45. 根據申請專利範圍第4 3項之醫藥組合物，其中該骨病為過份的骨質流失。 46. 根據申請專利範圍第4 5項之醫藥組合物，其中該骨病係選自下列所成組合之中者：骨疏鬆病，柏哲德氏骨病，高鈣血症，副甲狀腺官能過旺症，類固醇誘發貧骨症，風濕性關節炎所致骨質流失，骨髓炎所致骨質流失，溶 -20- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐)六、 Applicable patent Fan Yuan Trp Lys His Gin Asn Arg Asp Gin Glu Met Val Lys Lys lie lie Gin 260 265 270 Asp lie Asp Leu Cys Glu Ser Ser Val Gin Arg His Leu Gly His Ser 275 280 285 Asn Leii Thr Thr Glu Gin Leu Leu Ala Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys lie Ser Pro Glu Glu lie Glu Arg Thr Arg Lys Thr Cys Lys 305 310 315 320 Ser Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335 Gly Asp Gin Asp Thr Leu 'Lys Gly Leu Met Tyr Ala Leu Lys His Leu 340 345 350 Lys Thr Ser His Phe Pro Lys Thr Val Thr His Ser. Leu Arg Lys Thr 355 360 365 Met Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu 370 375 380 Phe Leu Glu Met Gle Asly Gin Val Gin Ser Val Lys lie Ser Cys 385 '390 395 400 Leu SEQBDNO: 125: Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie 15 10 15 Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr L'eu His Tyr Asp * 20 25 30 Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65-70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu ** 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 -14- This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm) 1221482 A8 B8 C8 D8, patent scope Leu Glu lie Glu Phe Cya Leu Lys His Arg Ser Cya Pro Pro Gly Phe 115 120 125 31y Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 ** 150 155 160 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys 165 170 175 Gly Asn Ala- Thr His Asp Asn lie Cys Ser Gly Asn Ser Glu Ser Thr ... 180 185 190 Gin Lvs Cvs Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg One 195 * 200 205 Phe Ala Val Pro Thr'Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 ^ 215 220 Asp Asn Leu Pr o Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 225 230 235 240 Lys Arg Gin His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255 Trp Lys His Gin Asn Lys Ala Gin Asp lie Val Ly.s Lys lie lie Gin 260 265 270 Asp lie Asp Leu Cys Glu Asn Ser Val Gin Arg His lie Gly Kis Ala 275 280 285 Asn Leu Thr Phe Glu Gin Leu Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys Val Gly Ala Glu Asp lie Glu Lys Thr lie Lys Ala Cys Lys 305 310 315 320 Pro Ser Asp Gin lie Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 325 330 335 Gly Asp Gin Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser 340 345 350 Lys Thr Tvr His Phe Pro Lys Thr Val Thr Gin -.Ser Leu Lys .Lys Thr 355. 360 365 lie Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leil 370 375 380 Phe Leu Glu Met He Gly Asn Gin Val Gin Ser Val Lys lie Ser Cya 385 390 395 400 O Leu -15- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) D8 VI. Patent Application Scope17. According to the patent application Wai polypeptide of Item 16, which is mammalian OPG. 18. A polypeptide according to item 17 of the scope of patent application, which is human PG. 19. The polypeptide according to item 16 of the scope of patent application, which is substantially free of other human proteins. 20. The polypeptide according to item 16 of the scope of the patent application, which has an amino acid sequence of residues 22-4001 (including residues 22 and 4001) shown in SEQ ID NO: 125. 21. A polypeptide according to item 16 of the scope of patent application, which has an amino acid sequence of residues 32-401 (including residues 32 and 401) shown in SEq id NO: 125. 22. The polypeptide according to item 16 of the scope of patent application, characterized in that it is a product of the expression of a foreign DNA sequence. 23. The polypeptide according to item 22 of the application, wherein the DNA is cDNA, genomic DNA or synthetic DNA. 24. The polypeptide according to item 16 of the scope of patent application, which has been modified by a water-soluble polymer. 25. The polypeptide according to item 24 of the application, wherein the water-soluble polymer is polyethylene glycol. 26. A polypeptide comprising: an amino acid sequence having at least about 164 amino acids, including four enrichments having characteristics of a cysteine-rich domain contained in the extracellular region of a tumor necrosis factor receptor A cysteine region, wherein the amino acid sequence is a part or all of the amino acid sequence shown in Se Q ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 125; and -16- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 6. Apply for a patent Fan Yuanyi to increase bone density. 27. A polypeptide having the amino acid sequence shown in SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 125, which has an amine end at residue 22, and 1 to 216 thereof The amino acid is deleted from the carboxy terminus. 28. The polypeptide according to item 27 of the patent application scope, which includes residues 22-185 (including residues 22 and 185), 22-189 (including 22 and 189), 22-194 (including residues 22 and 194), or 22 -201 (including residues 22 and 201) amino acid sequences. 29. The polypeptide according to item 28 of the scope of patent application, which further includes an Fc region contained in human IgG1 extended from the carboxyl terminal. 30 · — a polypeptide having the amino acid sequence shown in SEQ ID NO: 121, SEQ ID NO: 123, or SEQ ID NO: 125 and having an amino terminal at residue 22, wherein the amino terminal is deleted from the amino terminal Drop 1 to 10 amino acids. 31. The polypeptide according to item 30 of the patent application scope, which further deletes 1 to 216 amino acids from the carboxy terminus. 32. The polypeptide according to item 30 of the scope of patent application, which has residues 27-1 85 (including residues 27 and 185), 27-189 (including residues 27 and 189), and 27-194 (including residue 27 And 194), 27-401 (including residues 27 and 401), or 32-401 (including residues 32 and 401) amino acid sequences. 33. The polypeptide according to item 32 of the scope of patent application, which further includes an Fc region contained in human IgG1 extending from its basal end. 34. A polypeptide selected from the group consisting of: huOPG [22-201] -Fc huOPG [22-401] -Fc -17- This paper applies Chinese National Standard (CNS) A4 specifications ( (210 X 297 mm) 1221482 A8 B8 C8 D8 VI. Patent Application Range huOPG [22-180] -Fc huOPG met [22-401] -Fc huOPG Fc-met [22-201] huOPG met [22-185] huOPG met [22-189] huOPG met [22-194] huOPG met [27-185] huOPG met [27-189] huOPG met [27-194] huOPG met [32-401] huOPG met-lys [22-401] huOPG met [22-401] huOPG met [22-401] -Fc (P25A) huOPG met [22-401] (P25A) huOPG met [22-401] (P26A) huOPG met [22-401] (P26D) huOPG met [22-194] (P25A) huOPG met [22-194] (P26A) huOPG met met- (lys) 3 [22-401] huOPG met met-arg-gly-ser- (his) 6 [22-401 ] j wherein huOPG contains the amino acid sequence 歹 ij shown in SEQ ID NChl25. 35. A nucleic acid encoding a polypeptide according to item 34 of the scope of the patent application. 36. —A method to evaluate the ability of candidate substances to bind to OPG, its package-18- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1221482 A8 B8 C8 _ D8 I. Application scope — Including the use of OPG according to the scope of patent application No. 3, 16, 26, 27, 30 or 34 and the candidate substance is cultivated under conditions that can promote binding; and the substance being measured is measured. 37. A pharmaceutical composition for regulating the content of OPG in an animal, which contains a therapeutically effective amount of a nucleic acid according to item 1 of the scope of the patent application. 38. The pharmaceutical composition according to item 37 of the application, wherein the nucleic acid can contribute to an increase in tissue OPG level. 39. The pharmaceutical composition according to item 38 of the application, wherein the animal is a human. 40 · —A pharmaceutical composition for treating bone diseases, which comprises a therapeutically effective amount of a polypeptide according to the scope of patent application No. 3, 16, 26, 27, 30, or 34 and a pharmaceutically acceptable carrier, adjuvant, and solution Agents, stabilizers and / or antioxidants. 4L The composition according to item 40 of the patent application park, wherein the oPG is human OPG. 42. The scope of the patent application of the composition of 41, wherein the 〇pG having the amino acid sequence of 々 shown, the scope of the patent 79Q 810 830 AAAGTGAATGCCGAGAGTGTAGAGAGGATAAAACGGAGACACAGCTCACAAGAGCAAACC KVNAESVERIKRRHSSQEQT 850 870 890 TTCCAGCTGCTGAAGCTGTGGAAACATCAAAACAGAGACCAGGAAATGGTGAAGAAGATC FQ * Lr.LKLWKHQNRDQEMVKKI 910 930 950 ATCCAAGACATTGACCTCTGTGAAAGCAGCGTGCAGCGGCATCTCGGCCACTCGAACCTC IQDIDLCESSVQRHLGHSiiL 970 990 1010 ACCACAGAGCAGCTTCTTGCCTTGATGGAGAGCCTGCCTGGGAAGAAGATCAGCCCAGAA TTEQLLALHESLPGKKISPE -1030 1050 ·· 1070 GAGATTGAGAGAACGAGAAAGACCTGCAAATCGAGCGAGCAGCTCCTGAAGCTACTCAGT EI ^ ERTRKTCKSS EQLLKLLS ^ 1090 1110 1130 TTATGGAGGATCAAAAATGGTCACCAAGACACCTTGAAGGGCCTGATGTATGCCCTCAAG LW RIKNGDQDTLKGLMYALK 1150 1170 1190 CACTTGAAAACATCCCACTTrCCCAAAACTGTCACCCACAGTCTGAGGAAGACCATGAGG HLKTSHFPKTVTHSLRKTMR 1210 1230 1250 TTCCTGCACAGCTTCACAATGTACAGACTGTATCAGAAGCTCTTTTTAGAAATGATAGGG FLH. SFTMYRLYQ KLFLEMI G 1270 1290 1310 AATCAGGTTCAATCCGTGAAAATAAGCTGCTTATAACTAGGAATGGTCACTGGGCTGTTT NQVQSVK ISCL 43. A pharmaceutical composition for treating bone diseases, which contains a therapeutically effective amount of a polypeptide according to item 16 of the scope of patent application. 44. The pharmaceutical composition according to item 43 of the scope of patent application, wherein the polypeptide is human OPG. 45. The pharmaceutical composition according to item 43 of the scope of patent application, wherein the bone disease is excessive bone loss. 46. The pharmaceutical composition according to item 45 of the scope of patent application, wherein the bone disease is selected from the group consisting of: osteoporosis, Berger's bone disease, hypercalcemia, parathyroid function Prosperity, Steroid-induced anemia, Bone loss caused by rheumatoid arthritis, Bone loss caused by osteomyelitis, soluble -20- This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm)

六、申請專利範園骨性轉移及齒骨膜骨質流失。 47.根據申請專利範圍第4 3項之醫藥組合物，其芟、厶由 > 人匕括給用 >口療有效里的選自下列所成組合之中的物質：骨开3 ^ 育性蛋白質BMP-i至bmP-12，TGF-召族員，n 了怒發 W ’ TNF α抑制劑，副甲狀腺激素和其類似物，副甲」激素相關性蛋白質和其類似物，Ε系***素，錐^腺絲 1 η* , 1:膦酸鹽’及骨增強性礦物質。 48. —種由根據申請專利範圍第3、16、26、27、3〇或34项之促骨堆積肽單體聯合所形成之促骨堆積肽二體物。/、 49·根據申請專利範圍第4 8項之二體物，其係由鏈間雙硫所形成者。 50·根據申請專利範圍第4 8項之二體物，其係經由缔合衍生自人類IgGl的Fc區所形成的。 51. 根據申請專利範圍第4 8項之二體物，其基本上不含促骨堆積肽單體物和不具活性的二體物。 52. 根據申請專利範圍第4 8項之二體物，其中該單體物包括 SEQ ID NO : 125所示殘基22-401的胺基酸序列或其衍生物。 53.根據申請專利範圍第4 8項之二體物，其中該單體物包括 SEQ ID NO : 125所示殘基22-194的胺基酸序列。 -21- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐)6. Patent application Fan Yuan Bone metastasis and osteoperiosteal bone loss. 47. The medicinal composition according to item 43 of the scope of the patent application, wherein 芟, 厶 & > administered by humans > effective in oral therapy, selected from the group consisting of: bone open 3 ^ Sex proteins BMP-i to bmP-12, TGF-callers, anger W 'TNF α inhibitors, parathyroid hormones and their analogs, parathyroid hormones related proteins and their analogs, E-type prostate , Cone ^ glandular silk 1 η *, 1: phosphonate 'and bone enhancing minerals. 48. A osteopeptide dimer formed by combining osteopeptide monomers according to item 3, 16, 26, 27, 30 or 34 of the scope of the patent application. /, 49. According to the 48th bis-body of the scope of patent application, it is formed by interchain disulfide. 50. The dimer according to item 48 of the scope of patent application, which is formed by association of an Fc region derived from human IgG1. 51. According to item 48 of the application, the dimer is substantially free of bone accumulation peptide monomers and inactive dimers. 52. The dimer according to item 48 of the application, wherein the monomer includes the amino acid sequence of residues 22-401 shown in SEQ ID NO: 125 or a derivative thereof. 53. The dimer according to item 48 of the application, wherein the monomer includes the amino acid sequence of residues 22-194 shown in SEQ ID NO: 125. -21- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)