TW200525028A

TW200525028A - Nucleic acid construct encoding hirudin and methods for producing hirudin using a transgenic mammal whose genome comprises the nucleic acid construct

Info

Publication number: TW200525028A
Application number: TW93137499A
Authority: TW
Inventors: Ching-Fu Tu; Chon-Ho Yen; Chi-Kai Yang; I-Chung Cheng; Chich-Sheng Lin
Original assignee: Animal Technology Inst Taiwan
Priority date: 2003-12-03
Filing date: 2004-12-03
Publication date: 2005-08-01

Abstract

The present invention relates to a nucleic acid construct encoding hirudin and a method for preparing hirudin using transgenic mammals whose genome comprise the nucleic acid construct.

Description

200525028 九、發明說明：【發明所屬之技術領域】本發明係關於一種核酸構築，包括以可操作之方式結人的路蛋白基因啟動子、信號序列和編碼水虫至素的聚核菩㉟片段。本發明亦關於一種使用基因體含該核酸構築體之美因轉殖哺乳動物以產製水蛭素之方法。【先前技術】水蛭素是由65至66個胺基酸所組成的多胜肽（D〇dt，】等人，1986，FEBS Lett.202(2):373-7)，具有抗凝血的活性，其天然的水經素分離自歐洲醫蛭㈣心·❿d以）的唾腺’已知有三種水蛭素變體HV1、HV2和HV3，其彼此在胺基酸數目和蛋白質結構方面稍有不同。水蛭素可專一地結合凝血酶，抑制凝血酶的凝固活性。因此，水蛭素可用來治療與凝血酶之凝固活性有關的疾病，或預防、減輕或改善疾病的症狀，包括急性冠狀徵候群（acute c〇r〇nary syndr〇mes) (Weitz，J.I.和 Bates，E.R.，2003，Cardiovasc. Toxical·， 3(1):13-25) 〇在早期’可從歐洲醫蛭的唾液腺中純化和分離而獲得水虫至素。然而，藉著這類方法不易獲得足供醫學用途之含量的水虫至素。雖然亦可藉者使用基因重組技術，在原核生物表現系統（如大腸桿菌）中產製水蛭素，其申將水蛭素分泌至近膜間隙’再純化回收水蛭素，然而此種方法無法避免需瓦解細菌細胞再純化而導致降低水蛭素的產量。此外，原核生物系統對於所表現的多胜肽，缺乏轉譯後修飾。在這 200525028 類原核生物系統中產生之水蛭素的生物活性並不如預期。即使以酵母菌表現系統生產，水蛭素的產量和生物活性仍 ’、、：不南（美國專利第5,866,399號，·以及Courtney，M.等人 1989, Semin Thromb Hemost., 15(3):288-292) 〇從上文得知’對於發展有關水蛭素之量產方法，且所產生之水虫至素是可輕易回收的表現系統，仍有其必要性。【發明内容】在一方面，本發明提供一種核酸構築，包括以可操作之方式結合的酪蛋白基因啟動子、信號序列和編碼水虫至素的聚核甞酸片段。在另一方面，本發明提供一種產製水蛭素之方法，其步驟包括提供一種基因體含本發明之核酸構築之基因轉殖哺乳動物；從該哺乳動物收集其體液、分泌液或產物；以及從該等體液、分泌液或產物回收水經素。在另一方面，本發明提供一種產製水蛭素之方法，其步驟包括從基因體含本發明之核酸構築之基因轉殖哺乳動物分離組織或細胞；在適合表現水蛭素之條件下培養該細胞或組織；以及從該組織或細胞回收水蛭素。在另一方面，本發明提供一種表現載體，其包括本發明之核酸構築及複製起點。在另一方面，本發明提供一種包括本發明之表現載體之轉形的哺乳動物細胞或組織。在另一方面，本發明提供一種產製水蛭素之方法，其步驟包括在適合表現水蛭素之條件下培養本發明之經轉形的 95891.doc 200525028 哺乳動物細胞或組織，以及從該細胞或組織回收水虫至素。【實施方式】 I.定義本文使用之「構築」乙詞意指包括一或多個元件的核酸分子，例如編碼為所欲生產之蛋白質的聚核苷酸片段，以及驅動該蛋白質表現的啟動子。本文使用之「以可操作之方式結合」、「按照可操作之順序」和「以可操作之方式連接」等用語意指以這種方式連結的聚核苷酸元件，使該核酸分子得以容許特定基因的轉錄，及/或產生想要之蛋白質分子。該等用語亦意指以這種方式連結的胺基酸序列，得以產生具有功能的蛋白質。本文使用之「基因」乙詞意指一聚核甞酸片段，其編碼產製多胜肽或前驅物所必要的序列。可由全長的密碼序列或密碼序列的任何部分來編碼該多胜肽，只要仍保留想要的生物活性即可。本文使用之「水蛭素」乙詞意指任何形式的水蛭素或其類似物，天然經過分離的或人工合成的，只要仍保留想要的生物活性即可。本文使用之「表現載體」乙詞意指能夠攜帶並運送標的之核酉义片段至宿主細胞内，以表現該核酸片段之核酸分子。特疋而&，在重組DNA技術中使用的表現載體為質體、點接質體或病毒。本文使用之「宿主細胞」乙詞意指宿主的細胞，其可利用載體（如質體）轉形或轉殖之。 95891.doc 200525028 本文使用之「非人類哺乳動物」乙詞意指任何非人類的哺乳動物，其基因體可含本發明之核酸構築。這類非人類哺乳動物包括，但不限於，嚙齒動物、非人類的靈長類、豬、牛、山羊、綿羊、反离動物、兔類（lag〇m〇rphs)、馬。本文使用之「轉殖基因」乙詞意指外來的基因，其可導入胚胎幹細胞、新受精的卵或早期的胚中，藉此置入生物體内。根據本發明，轉殖基因意指編碼水蛭素或其類似物的基因或聚核甞酸片段。本文使用之「啟動子元件」或「啟動子」乙詞意指在dna 聚合物中，位在基因5|端（即上游）的DNA序列，並提供基因開始轉錄成mRNA的位置。 II·本發明之目的 A·核酸構築在一方面，本發明提供一種核酸構築，包括以可操作之方式結合的赂蛋白基因啟動子、信號序列和編碼水虫至素的聚核苷酸片段。 μ 本發明之核酸構築所含赂蛋白基因啟動子可分離自哺乳動物之酪蛋白基因’該等哺乳動物包括，但不限於，人類、豬、牛、馬、山羊、錦羊、駱乾或响齒動物。在一具體實施例中’本發明核酸構築所含路蛋白基因啟動子係:山苹之卜酪蛋白基因。許多商業上可獲得之載體，例如，購自200525028 IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to a nucleic acid construct, including a operon gene promoter, a signal sequence, and a polynuclear polynuclear fragment encoding a water insect toxin. The present invention also relates to a method for producing hirudin by transgenic mammals using a gene construct containing the nucleic acid construct. [Prior art] Hirudin is a polypeptide composed of 65 to 66 amino acids (Dodt, et al., 1986, FEBS Lett. 202 (2): 373-7), and has anticoagulant properties. Activity, its natural hydrolysate is isolated from the salivary glands of the European Medical Care Center, and there are three known hirudin variants HV1, HV2 and HV3, which are slightly different from each other in the number of amino acids and protein structure different. Hirudin specifically binds thrombin and inhibits the coagulation activity of thrombin. Therefore, hirudin can be used to treat diseases related to the coagulation activity of thrombin, or to prevent, reduce or improve the symptoms of the disease, including acute coronary syndromes (Weitz, JI, and Bates, ER, 2003, Cardiovasc. Toxical ·, 3 (1): 13-25) ○ In the early stage, it was possible to purify and isolate salivary glands from European medicinal plants to obtain water insects. However, it is not easy to obtain water insecticides sufficient for medical purposes by such methods. Although you can also use genetic recombination technology to produce hirudin in a prokaryotic expression system (such as E. coli), it claims to secrete hirudin to the near-membrane space and then purify and recover hirudin. However, this method cannot avoid disintegrating bacteria. Cell repurification results in reduced hirudin production. In addition, prokaryotic systems lack posttranslational modification of the expressed polypeptides. The biological activity of hirudin produced in this 200525028-type prokaryotic system is not as expected. Yield and biological activity of hirudin even when produced by yeast expression system ', :: Bhutan (US Patent No. 5,866,399, and Courtney, M. et al. 1989, Semin Thromb Hemost., 15 (3): 288 -292) 〇 From the above, it is known that 'there is still a need for the development of a method for mass production of hirudin, and the produced water insects are a performance system that can be easily recovered. [Summary of the Invention] In one aspect, the present invention provides a nucleic acid construct comprising a casein gene promoter, a signal sequence, and a polynucleic acid fragment that encodes aquatic hormones in an operable manner. In another aspect, the present invention provides a method for producing hirudin, the steps of which include providing a transgenic mammal whose gene body contains the nucleic acid construct of the present invention; collecting body fluid, secretion, or product from the mammal; and Hydrocycline is recovered from such body fluids, secretions or products. In another aspect, the present invention provides a method for producing hirudin, comprising the steps of isolating a tissue or cell from a transgenic mammal whose gene body contains the nucleic acid construct of the present invention; and culturing the cell under conditions suitable for expressing hirudin. Or tissue; and recovering hirudin from the tissue or cell. In another aspect, the present invention provides a performance vector comprising a nucleic acid construct and a replication origin of the present invention. In another aspect, the invention provides a transformed mammalian cell or tissue comprising the expression vector of the invention. In another aspect, the present invention provides a method for producing hirudin, comprising the steps of culturing the transformed 95891.doc 200525028 mammalian cell or tissue of the present invention under conditions suitable for expressing hirudin, and from the cell or Tissues recover water insects. [Implementation] I. Definition The term "construction" as used herein means a nucleic acid molecule including one or more elements, such as a polynucleotide fragment encoding a protein to be produced, and a promoter driving the expression of the protein . As used herein, the terms "combined in an operable manner", "in an operable order", and "operably connected" mean the polynucleotide elements linked in such a way that the nucleic acid molecule is allowed to Transcription of specific genes and / or production of desired protein molecules. These terms also mean that the amino acid sequences linked in this way are capable of producing a functional protein. As used herein, the term "gene" means a polynucleic acid fragment that encodes a sequence necessary for the production of a polypeptide or precursor. The polypeptide can be encoded by the full-length code sequence or any portion of the code sequence, as long as the desired biological activity is still retained. The term "hirudin" as used herein means any form of hirudin or an analogue thereof, naturally isolated or artificially synthesized, so long as the desired biological activity is retained. As used herein, the expression "expression vector" means a nucleic acid molecule capable of carrying and transporting a target nuclear sense fragment into a host cell to express the nucleic acid fragment. In particular, &, the expression vectors used in recombinant DNA technology are plastids, aptamers or viruses. As used herein, the term "host cell" means a host cell that can be transformed or transplanted using a vector, such as a plastid. 95891.doc 200525028 The term "non-human mammal" as used herein means any non-human mammal whose genome can contain the nucleic acid construct of the present invention. Such non-human mammals include, but are not limited to, rodents, non-human primates, pigs, cattle, goats, sheep, anti-ionizing animals, lagomorphs, and horses. The term "transgenic gene" as used herein means a foreign gene that can be introduced into an embryonic stem cell, a newly fertilized egg, or an early embryo, thereby being inserted into an organism. According to the present invention, a transgenic gene means a gene or a polynucleic acid fragment encoding hirudin or an analog thereof. As used herein, the term "promoter element" or "promoter" means a DNA sequence located at the 5 | end (ie, upstream) of a gene in a DNA polymer and provides a position for the gene to begin transcription into mRNA. II. Objects of the present invention A. Nucleic acid construct In one aspect, the present invention provides a nucleic acid construct comprising a bridging protein gene promoter, a signal sequence, and a polynucleotide fragment encoding a water insectin in an operable manner. μ The promoter of the bridging protein gene contained in the nucleic acid construct of the present invention can be isolated from the casein gene of mammals. These mammals include, but are not limited to, human, pig, cattle, horse, goat, brom Tooth animal. In a specific embodiment, the promoter system of the road protein gene contained in the nucleic acid construct of the present invention is the apple casein gene. Many commercially available vectors, for example, purchased from

Invitr〇gen公司之pBcl載體’可提供適當的酿蛋白基因啟動子，以構築本發明之核酸構築。本文使用之「彳έ號序列」意指一胺基酸序列或其對應之 95891.doc 200525028 核甘酸序列’其可決定以可操作之方式結合至該信號序列之多胜肽的表現位置。已知信號序列在重組蛋白質分泌至礼汁之技術中扮演重要角色（Persuy，Μ· Α·等人，1995, 165(2)·· 291-6)。根據本發明，使用信號序列可提昇水虫至素從乳腺細胞分泌至該等細胞所分泌之乳汁中。本發明所使用之信號序列可衍生自哺乳動物之酪蛋白基因，該等哺乳動物包括，但不限於人類、豬、牛、山羊、綿羊、馬、駱駝或嚙齒動物。在一具體實施例中，本發明所使用之信號序列係衍生自山羊之卜酪蛋白基因，較佳為seqidn〇: 9所示之具45個核：y：酸之序列。先前技藝已詳細描述天然水蛭素及對應基因之序列内容，例如，GeneBank所示編號為M12693之序列（SEQ m NO: 15)。本發明所使用之水蛭素之胺基酸序列可含有一或多個胺基酸殘基之刪除、新增或取代，其係導致沉默改變，因而實質上不影響水虫至素之活性。亥項技術者可從前案資料獲得水蛭素之核甞酸序列，以製得編碼水蛭素之聚核芬酸片段，供製得本發明之核酸構築。此一領域已發展純熟的遺傳工程方法，如DNA選殖及載體建構，可參見例如，Current PrQtQeQls inInvitrogen's pBcl vector ' can provide an appropriate promoter of the gene for brewing protein to construct the nucleic acid construct of the present invention. As used herein, the "sequence number" means a monoamino acid sequence or its corresponding 95891.doc 200525028 nucleotide sequence 'which can determine the expression position of a peptide that is operatively bound to the signal sequence. It is known that the signal sequence plays an important role in the technology of recombinant protein secretion into ceremonial juice (Persuy, M.A. et al., 1995, 165 (2) .. 291-6). According to the present invention, the use of a signal sequence can promote the secretion of water insects from the breast cells into the milk secreted by these cells. The signal sequence used in the present invention may be derived from the casein gene of mammals. Such mammals include, but are not limited to, humans, pigs, cattle, goats, sheep, horses, camels or rodents. In a specific embodiment, the signal sequence used in the present invention is derived from a goat casein gene, preferably a sequence with 45 cores: y: acid as shown by seqidn 0: 9. The sequence contents of natural hirudin and corresponding genes have been described in detail in the prior art, for example, the sequence numbered M12693 (SEQ m NO: 15) shown in GeneBank. The amino acid sequence of hirudin used in the present invention may contain deletions, additions or substitutions of one or more amino acid residues, which results in silent changes, and thus does not substantially affect the activity of the hydrozoan-toxin. Those skilled in the art can obtain the nucleotide sequence of hirudin from the data of the previous case to obtain a polynuclear fenamic acid fragment encoding hirudin, which can be used to prepare the nucleic acid construct of the present invention. Skilled genetic engineering methods have been developed in this area, such as DNA selection and vector construction, see for example, Current PrQtQeQls in

Molecular Biology ( Frederick Μ· A.等人，2〇〇lMolecular Biology (Frederick M.A. et al., 2001

John Wiley & Sons，Inc·)。可使用習知的遺傳工程方法將前述元件以適當順序連結起來，以製得本發明之核酸構築。製備本發明之核酸構築之詳細流程說明於下文之實例中。 95891.doc -10· 200525028 在-具體實施例中’本發明之核酸構築更包括—或多個 ZML球蛋白絕緣子（/3_globin insuIat〇r)元件。已有前案資料報導/ML球蛋白絕緣子元件可使一轉殖基因免於受到染色體位置影響（Chung，j. η.等人，1997，Pr〇c Nat〗 Acad 如亂，94(2)·· 575，以及 Chung，； H 等人，1993,㈣， 74(3). 5G5-14)。在本發明中，建議可使用^乳球蛋白絕緣子元件以提昇本發明之核酸構築在插人於哺乳動物之基因體時之穩定度。在另-具體實施例中，本發明之核酸構築所含路蛋白基因啟動子可置換為另—啟動子，其係分離自以下所組成之核酸片段或基因：哺乳動物之酸性乳清蛋白基因、乳白蛋白基因及乳球蛋白基因，較佳為《•乳白蛋白（aLA)啟動子，該等哺乳動物包括，但不限於人類、豬、牛、山羊、綿羊、馬、駱銳、或P齒齒動物。因此在另方面，本發明提供另一種核酸構築，其包括aLA啟動子及編碼水蛭素之聚核苷酸片段。本發明之核酸構築可導入非人類哺乳動物之生殖細胞或受精印之基因體中，以產生非人類基因轉殖哺乳動物。本發明之核酸構築所含啟動子可使水經素表現於基因轉瘦哺乳動物之各種組織或細胞，較佳為乳腺組織或乳腺細胞，或使水經素產生於基因㈣哺乳動物之體液或分泌液中，較佳為乳汁、血液、尿液、及***，更佳者為乳汁。 B·基因轉殖動物在另-方面，本發明提供非人類之基因轉殖動物，其基 95891.doc 200525028 因體包括本發明之核酸構築。近年來分子遺傳學已提供有力工具及方法，以便將有興 I之基因導入非人類哺乳動物之基因體中而產生基因轉殖動物’其可用於研究人類疾病或產製想要的物質。一般而吕，可選擇在各種發育階段之胚作為導入有興趣之基因之 ‘的此一領域已提供各種類胚之發育階段而將有興趣之基因導入至胚細胞之方法。對於顯微注射方法而言，可使用原核胚作為導入有興趣之基因之標的。藉由這樣的手段，注射於胚之基因可在該胚第一次***之前進入該胚之基因體内。如此一來，由該胚衍生之動物之所有細胞均可 T有所導入之基因。此一領域亦已提供許多文獻說明產製轉殖基因動物之技術，例如，Tu CF等人，int Surg· 1999; 84(2). 176_182 以及 TuCF 等人，Xen〇tranSpiantati〇n 2〇〇3; ^(5): 503。本發明之非人類基因轉殖哺乳動物可由顯微注射方法製得。在本發明之具體實施例中，供胚者係經有效量之孕馬血清***（PMSG)及人類絨毛膜***（hCG ) 處理，繼而與雄性配種動物交配。將受精卵從輸卵管沖出，然後將本發明之核酸構築注射至原核胚。將存活之胚移至 ***受胚母體内，便可生出本發明之基因轉殖動物。本發明之非人類基因轉殖哺乳動物包括，但不限於，豬、牛、馬、山羊、•絡騎、綿羊或喃齒動物。本發明之基因轉殖動物之基因體可由習知方法鑑定，例如，PCR或南方墨潰分析方法。 95891.doc -12- 200525028 此：領域已發展純熟的基因轉殖技術及分析基因轉殖動物之基因體之技術。可使用習 ^ 0 ., 白知的基因轉殖技術製得本 4明《基因轉殖動物，並使用習知的分析方法鑑定基因轉殖動物之基因體。製備本發明之基因轉殖動物及分析其基因體之詳細流程係說明於下文之實例中本發明之基因轉殖動物所含之轉瘦基因係穩线嵌入复生殖種系細胞中。理論上約有抓之子代可從其親代獲得該轉殖基因，亦有可能因轉殖基因之以嵌合體（m〇Saic)或多點钱插至基因轉殖動物基因體中之不同染色體，致其第一子代㈣之傳代率，少於或多於5G%。在—具體實施例中，本發明之基因轉殖動物之生殖種系傳代率（基因體含該轉殖基因之第-代子代（F1)之數目/第_代子代（F1)之總數達近50%。本發明之基因轉殖動物或其子代可於敎組織或細胞表現水蛵素’較佳為乳腺組織或乳腺細胞，或於各種體液、分泌液或產物中產生水蛭素，較佳為乳汁、血液、尿液、及***，更佳為乳汁。在-具體實施例中，本發明之基因轉殖動物或其子代可從其乳腺分泌含水蛭素之乳汁。從本發明之基因轉殖動物或其子代產生之含水蛭素之乳汁展現出高度的抗凝集活性，例如，每微升之乳汁可達⑺丨至的抗凝血酶單位或更高濃度之活性。且，該乳汁中之水蛭素的抗凝集活性持續整個泌乳期間。 95891.doc 13 200525028 c·使用本發明之基因轉殖動物產製水蛭素之方法因此，在另一方面，本發明提供一種產製水蛭素之方法，其步驟包括提供本發明之基因轉殖哺乳動物、從該哺乳動物收集其體液、分泌液或產物以及從該等體液或分泌液回收水蛭素。較佳地，本發明之方法係收集基因轉殖哺乳動物之乳汁、血液、尿液、及***。更佳地，本發明之方法係收集基因轉殖哺乳動物之乳汁，該乳汁展現出高度的抗血液凝集活性，例如，每微升之乳汁達01至4〇抗凝血酶單位或更高濃度之酵素活性，且該乳汁中之水蛭素的抗凝集活性持續該基因轉殖哺乳動物之整個泌乳期間。此一領域已揭示各種純化水蛭素之方法。可使用適當方法，例如，親和性管柱層析法或高效能液相層析法（hplc)，從本發明之基因轉殖動物之體液或分泌液中純化水蛭素。如前所述，本發明之基因轉殖動物或其子代可於特定組織或細胞表現水蛭素。因此，在另一方面，本發明提供一種產製水蛭素之方法，其步驟包括從本發明之基因轉殖動物分離組織或細胞，在適合表現水蛭素之條件下培養該細胞或組織，以及從該組織或細胞回收水蛭素。在一具體實施例中，該方法係從本發明之基因轉殖動物分離乳腺組織或乳腺細胞。從動物分離組織或細胞以及在活體外培養該等組織或細胞之技術已為此一領域所熟知。可使用適當方法進行動物組織或細胞之分離，並在適當條件下於活體外培養該等組織或細胞。從動物組織或細胞分離及純化水蛭素之技術亦 95891.doc -14- 200525028 之親和性管柱層析法或高組織及藉此產製水蛭素之為此一領域所熟知，例如，前述效能液相層析法（HPLC)。 D·表現載體、經其轉形之細胞或方法在另一方面，本發明接征—從士仏種表現載體，其包括如前所述之核酸構築及複製起點。根據本發明，該複製起點係使· 該表現載體得以在哺乳動物細胞（較佳為乳腺細胞）中複製< 並表現所攜帶之基因產物。各種可使核酸表現於哺乳動物細胞之複製起點為此一領域所習知。 _ 本發明之表現載體係用於在嚼乳動物細胞中表現水虫至素’其所含核酸構築之元件（例如，赂蛋白基因啟動子或— 啟動子、彳5號序列、編碼水蛭素之聚核甞酸片段、及一或多個心乳球蛋白絕緣子）已說明於上文中。較佳地，本發明之表現載體更包括一篩選標記。更佳地，本發明之表現載體更包括一標籤序列，因此，可產生一融合多核苷酸，而有利於後續之純化程序。此一領域已發展純熟的遣傳工程方法，如前述之鲁 Current Protocols i n Molecular Biology (Frederick Μ·Α·等人，2001· John Wiley & Sons，John Wiley & Sons, Inc.). The aforementioned elements can be linked in an appropriate order using a conventional genetic engineering method to prepare a nucleic acid construct of the present invention. Detailed procedures for preparing the nucleic acid constructs of the invention are illustrated in the examples below. 95891.doc -10 · 200525028 In specific embodiments, the nucleic acid construct of the present invention further includes—or multiple ZML globulin insulator (/ 3_globin insuIatOr) elements. It has been reported in previous cases that the ML globulin insulator element can protect a transgenic gene from being affected by chromosomal position (Chung, j. Η. Et al., 1997, Proc Nat. Acad Ruran, 94 (2) · · 575, and Chung ,; H et al., 1993, ㈣, 74 (3). 5G5-14). In the present invention, it is suggested that the lactoglobulin insulator element can be used to improve the stability of the nucleic acid of the present invention when it is inserted into the mammalian genome. In another specific embodiment, the promoter of the road protein gene contained in the nucleic acid construct of the present invention can be replaced with another promoter, which is isolated from a nucleic acid fragment or gene composed of: the acidic whey protein gene of mammals, The lactalbumin gene and the lactoglobulin gene are preferably a lactalbumin (aLA) promoter. These mammals include, but are not limited to, humans, pigs, cattle, goats, sheep, horses, Luo Rui, or Ptooth animal. Thus in another aspect, the invention provides another nucleic acid construct comprising an aLA promoter and a polynucleotide fragment encoding hirudin. The nucleic acid construct of the present invention can be introduced into the germ cells or fertilized genomic bodies of a non-human mammal to produce a non-human gene transgenic mammal. The promoter contained in the nucleic acid structure of the present invention can cause meridin to be expressed in various tissues or cells of a gene-transformed mammal, preferably mammary gland tissue or mammary gland cells, or to cause meridin to be generated in the body fluid of a gene or mammal or Among the secretions, milk, blood, urine, and semen are preferred, and milk is more preferred. B. Gene Transgenic Animals In another aspect, the present invention provides non-human gene transgenic animals whose bases are 95891.doc 200525028 and include the nucleic acid constructs of the present invention. In recent years, molecular genetics has provided powerful tools and methods for introducing genes of interest I into the genomes of non-human mammals to produce transgenic animals, which can be used to study human diseases or produce desired substances. Generally, Lv can choose embryos at various developmental stages as the introduction of genes of interest. This area of ‘there has been provided a method of introducing genes of interest into embryonic cells at various stages of embryoid development. For microinjection methods, prokaryotes can be used as targets for the introduction of genes of interest. With such a method, the gene injected into the embryo can enter the gene of the embryo before the embryo divides for the first time. In this way, all cells of the animal derived from the embryo can have the introduced gene. Many literatures have also been provided in this field to explain the technology for producing transgenic animals, for example, Tu CF et al., Int Surg 1999; 84 (2). 176_182 and TuCF et al., Xen〇tranSpiantati〇n 2003. ; ^ (5): 503.; The non-human gene transgenic mammal of the present invention can be prepared by a microinjection method. In a specific embodiment of the present invention, the donor is treated with an effective amount of pregnant horse serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and then is mated with a male breeding animal. The fertilized egg is washed out of the fallopian tube, and then the nucleic acid construct of the present invention is injected into the prokaryotic embryo. By transferring the surviving embryo into the surrogate embryo, the transgenic animal of the present invention can be produced. Non-human genetically modified mammals of the present invention include, but are not limited to, pigs, cattle, horses, goats, horses, sheep, or squirrels. The gene body of the transgenic animal of the present invention can be identified by a conventional method, for example, PCR or Southern blot analysis. 95891.doc -12- 200525028 This: The field has developed sophisticated gene transfer technology and technology to analyze the gene body of gene transfer animals. The genetic transformation technology of Bai Zhi can be used to obtain the book 4 "Genetically Transplanted Animals", and the known analytic methods can be used to identify the gene bodies of genetically transgenic animals. The detailed procedure for preparing the transgenic animal of the present invention and analyzing its gene body is illustrated in the following examples. The stable line of the lean gene line contained in the transgenic animal of the present invention is embedded in the reproductive germline cells. Theoretically probable offspring can obtain the transgene from their parents. It is also possible that the transgene can be inserted into different chromosomes in the gene body of the transgenic animal due to chimera (moSaic) or multi-point money. As a result, the passage rate of the first offspring was less than or more than 5G%. In a specific embodiment, the generation rate of the germline of the transgenic animal of the present invention (the number of the first-generation progeny (F1) / the first-generation progeny (F1) of the gene body containing the transgenic gene) The total amount is nearly 50%. The transgenic animals or their progeny of the present invention can express hirudin in maggot tissues or cells, preferably breast tissue or breast cells, or produce hirudin in various body fluids, secretions or products. It is preferably milk, blood, urine, and semen, and more preferably milk. In a specific embodiment, the genetically modified animal of the present invention or its progeny can secrete the milk of hydratin from its breast. From this The milk containing hydrolysin produced by the transgenic animal of the invention or its progeny exhibits a high degree of anti-agglutination activity, for example, the antithrombin unit or higher concentration of activity per microliter of milk can be reached. And, the anti-agglutination activity of hirudin in the milk lasts throughout the lactation period. 95891.doc 13 200525028 c. Method for producing hirudin using the gene transgenic animal of the present invention Therefore, in another aspect, the present invention provides A method for producing hirudin, which The steps include providing a transgenic mammal of the present invention, collecting body fluids, secretions, or products from the mammal, and recovering hirudin from the body fluids or secretions. Preferably, the method of the present invention is collecting transgenic mammals Animal milk, blood, urine, and semen. More preferably, the method of the present invention collects the milk of a transgenic mammal, which milk exhibits high anti-hemagglutination activity, for example, up to 01 per microliter of milk To 40 antithrombin units or higher enzyme activity, and the anticoagulant activity of hirudin in the milk lasts throughout the lactation period of the transgenic mammal. This field has revealed a variety of purified hirudin. Method. Hirudin can be purified from the body fluid or secretion of the transgenic animal of the present invention using an appropriate method, for example, affinity column chromatography or high performance liquid chromatography (hplc). The gene transgenic animal or its progeny of the present invention can express hirudin in specific tissues or cells. Therefore, in another aspect, the present invention provides a method for producing hirudin, The steps include isolating a tissue or cell from a transgenic animal of the invention, culturing the cell or tissue under conditions suitable for the expression of hirudin, and recovering hirudin from the tissue or cell. In a specific embodiment, the method is from The genetically transgenic animal of the present invention isolates breast tissue or breast cells. Techniques for isolating tissues or cells from animals and culturing such tissues or cells in vitro are well known in the art. Appropriate methods can be used to perform Isolate and culture these tissues or cells in vitro under appropriate conditions. Techniques for isolating and purifying hirudin from animal tissues or cells are also 95891.doc -14- 200525028 affinity column chromatography or high tissue and borrow The production of hirudin is well known in this field, for example, the aforementioned performance liquid chromatography (HPLC). D. Expression vectors, transformed cells or methods thereof In another aspect, the present invention is directed to-from The expression vector includes a nucleic acid construct and a replication origin as described above. According to the present invention, the origin of replication enables the expression vector to replicate < and express the gene product carried in mammalian cells (preferably breast cells). Various origins of replication that allow nucleic acids to be expressed in mammalian cells are known in the art. _ The expression vector of the present invention is used for expressing elements of the watercepsin to its element contained in chewing milk cells (for example, the promoter of the bristle protein gene or — promoter, the sequence of # 5, the encoding of hirudin Nucleic acid fragments, and one or more pneumoglobulin insulators) have been described above. Preferably, the expression vector of the present invention further comprises a selection marker. More preferably, the expression vector of the present invention further includes a tag sequence. Therefore, a fusion polynucleotide can be generated, which is beneficial for subsequent purification procedures. This field has developed sophisticated methods for repatriation engineering, such as the aforementioned Current Protocols i Molecular Biology (Frederick M.A. et al., 2001. John Wiley & Sons,

Inc·)。可使用習知的遗傳工程方法將前述元件以適當順序連結起來，以製得本發明之表現載體。製 · 備本發明之表現載體之詳細流程說明於下文之實例中。本發明之表現載體可導入哺乳動物細胞或組織，以獲得可表現水經素之經轉形的哺乳動物細胞及組織。因此，在 95891.doc -15- 200525028 另-方面，本發明提供經前述表現載體轉形的哺乳動物細胞或組織’或直接/分離自哺乳動物之乳腺植織，其可用於表現水絰素。在-具體實施例中，本發明之轉形的嗜乳動物細胞為購自食品工業發展研究所（台灣，新竹）之細胞銀行的小鼠乳腺上皮細胞株NMuMG (BCRC60087)。可使用各種習知的轉形方法製得本發明之經轉形的哺乳動物細胞或組織，例如，氣化鈣處理方法、鈣_pEG方法、電轉殖方法、DEAE-葡聚糖或脂質體媒介的轉染方法以及顯微注射方法。此等轉形技術可參見如前所述之Inc.). The aforementioned elements can be linked in an appropriate order using a conventional genetic engineering method to prepare the expression vector of the present invention. The detailed process for preparing the expression vector of the present invention is illustrated in the examples below. The expression vector of the present invention can be introduced into mammalian cells or tissues to obtain transformed mammalian cells and tissues capable of expressing meridian. Accordingly, in 95891.doc -15-200525028, in another aspect, the present invention provides mammalian cells or tissues 'transformed with the aforementioned expression vectors' or directly / isolated from mammalian mammary glands, which can be used to express hirudin. In a specific embodiment, the transformed breast milk animal cell of the present invention is a mouse mammary epithelial cell line NmuMG (BCRC60087) purchased from Cell Bank of the Food Industry Development Institute (Hsinchu, Taiwan). Transformed mammalian cells or tissues of the present invention can be made using various conventional transformation methods, such as calcium vaporization methods, calcium-pEG methods, electrotransplantation methods, DEAE-dextran or liposome vehicles Transfection method and microinjection method. These transformation techniques can be found in the

Protocols in Molecular Biol〇gy(Frederick Μ·Α·等人，2001·Protocols in Molecular Biology (Frederick M.A. et al., 2001.

John Wiley & Sons，Inc·)。製備本發明之經轉形的哺乳動物細胞或組織之詳細流程係說明於下文之實例中。在另一方面，本發明更提供一種產製水蛭素之方法，其步驟包括在適合表現水蛭素之條件下培養本發明之經轉形的哺乳動物細胞或組織，以及從該細胞或組織回收水虫至素。如前所述，從動物組織或細胞純化水蛭素之技術已為此一領域所熟知，例如，親和性管柱層析法或高效能液相層析法（HPLC)。可使用適當方法萃取本發明之經轉形的組織或細胞，繼而分離並純化水蛭素。參考以下的貫施例將使本發明更為顯而易知。以下所說明的實施例僅係以解說方式提出，而非意圖對本發明做任何限制。實例1 ··編碼水蛭素之全長DNA片段的合成以在Genbank所示登錄編號為M12693之水蛭素基因序列 95891.doc -16· 200525028 (SEQ ID NO : 15)為基礎，設計四個單股的DNA片段， Hi-AF(SEQ ID NO ·· 1)、Hi-AR(SEQ ID NO : 2)、Hi-BF(SEQ ID NO : 3)和 Hi-BR(SEQ ID NO : 4)，其中 Hi-AF和 Hi-AR是彼此互補的，且Hi-BF和Hi-BR是彼此互補的（表1)。此外，根據前述四個DNA片段的5f-終端序列，設計四個引子 Hi-PCR-AF(SEQ ID NO ·· 5)、Hi-PCR-AR(SEQ ID NO : 6)、 Hi-PCR-BF(SEQ ID NO : 7)和 Hi-PCR-BR(SEQ ID NO ·· 8)(表 1)。 95891.doc 200525028John Wiley & Sons, Inc.). The detailed procedure for preparing the transformed mammalian cells or tissues of the present invention is illustrated in the examples below. In another aspect, the present invention further provides a method for producing hirudin, which comprises the steps of culturing the transformed mammalian cell or tissue of the present invention under conditions suitable for expressing hirudin, and recovering water insects from the cell or tissue. To prime. As mentioned earlier, techniques for purifying hirudin from animal tissues or cells are well known in this field, for example, affinity column chromatography or high performance liquid chromatography (HPLC). The transformed tissues or cells of the present invention can be extracted using an appropriate method, and hirudin can then be isolated and purified. Reference to the following examples will make the present invention more apparent. The embodiments described below are presented by way of illustration only and are not intended to limit the invention in any way. Example 1 Synthesis of a full-length DNA fragment encoding hirudin Based on the hirudin gene sequence 95891.doc -16 · 200525028 (SEQ ID NO: 15) shown in Genbank with accession number M12693, four single-stranded DNA fragments, Hi-AF (SEQ ID NO · · 1), Hi-AR (SEQ ID NO: 2), Hi-BF (SEQ ID NO: 3), and Hi-BR (SEQ ID NO: 4), where Hi -AF and Hi-AR are complementary to each other, and Hi-BF and Hi-BR are complementary to each other (Table 1). In addition, based on the 5f-terminal sequences of the aforementioned four DNA fragments, four primers Hi-PCR-AF (SEQ ID NO · 5), Hi-PCR-AR (SEQ ID NO: 6), and Hi-PCR-BF were designed. (SEQ ID NO: 7) and Hi-PCR-BR (SEQ ID NO. · 8) (Table 1). 95891.doc 200525028

表1 :水蛭素基因Ml2693之序列、DNA片段和引子水蛭素基因、 DNA片段和引子序列(5’-3’） SEQINNO M12693 ^一 ATGAAGGTCCTCATCCTTGCCTGTCTGGTGG CTCTGGCCATTGCAGTTGTTTACACCGACTG CACTGAATCCGGTCAGAACCTGTGCCTGTGC GAAGGCTCTAACGTTTGTGGCCAGGGCAACA AATGCATCCTGGGCTCTGACGGCGAAAAAAA TCAATGCGTTACTGGCGAAGGTACTCCGAAA CCGCAGTCTCACAACGACGGCGACTTTGAAG AAATCCCGGAAGAATACCTGCAATAA 15 Hi-AF 〜— gatcctttatggttgtttacactgactgcac tgaatccggtcagaacctgtgcctgtgcgaa ggctctaacgtttgcggccagggcaacaaat gcatcctgggc 1 Hi-AR ctctagagcccaggatgcatttgttgccctg gccgcaaacgttagagccttcgcacaggcac aggttctgaccggattcagtgcagtcagtgt aaacaaccataaag 2 Hi-BF tctagaggcgaaaaaaatcaatgcgttactg gcgaaggtactccgaaaccgcagtctcacaa cgacggcgactttgaagaaatcccggaagaa tacctgcaataatagggc 3 Hi-BR ggccgccctattattgcaggtattcttccgg gatttcttcaaagtcgccgtcgttgtgagac tgcggtttcggagtaccttcgccagtaacgc attgatttttttcgc 4 Hi-PCR-AF tcgggatcctttatggttgtttacactgact gc 5 Hi-PCR-AR gcctctagagcccaggatgcatttgttgccc 6 Hi-PCR-BF ggctctagaggcgaaaaaaatcaatgcgtta ctggcga 7 Hi-PCR-BR catgcggccgccctattattgcaggtattct t 8 95891.doc 18- 200525028Table 1: The sequence of the gene Ml2693 hirudin, DNA fragments and primers hirudin genes, DNA fragments and primer sequence (5'-3 ') SEQINNO M12693 ^ a ATGAAGGTCCTCATCCTTGCCTGTCTGGTGG CTCTGGCCATTGCAGTTGTTTACACCGACTG CACTGAATCCGGTCAGAACCTGTGCCTGTGC GAAGGCTCTAACGTTTGTGGCCAGGGCAACA AATGCATCCTGGGCTCTGACGGCGAAAAAAA TCAATGCGTTACTGGCGAAGGTACTCCGAAA CCGCAGTCTCACAACGACGGCGACTTTGAAG AAATCCCGGAAGAATACCTGCAATAA 15 Hi-AF ~- gatcctttatggttgtttacactgactgcac tgaatccggtcagaacctgtgcctgtgcgaa ggctctaacgtttgcggccagggcaacaaat gcatcctgggc 1 Hi-AR ctctagagcccaggatgcatttgttgccctg gccgcaaacgttagagccttcgcacaggcac aggttctgaccggattcagtgcagtcagtgt aaacaaccataaag 2 Hi-BF tctagaggcgaaaaaaatcaatgcgttactg gcgaaggtactccgaaaccgcagtctcacaa cgacggcgactttgaagaaatcccggaagaa tacctgcaataatagggc 3 Hi-BR ggccgccctattattgcaggtattcttccgg gatttcttcaaagtcgccgtcgttgtgagac tgcggtttcggagtaccttcgccagtaacgc attgatttttttcgc 4 Hi-PCR-AF tcgggatcctttatggttgtttacactgact gc 5 Hi-PCR-AR gcctctagagcccaggatgcatttgttgccc 6 Hi-PCR-BF ggctctagaggcgaa aaaaatcaatgcgtta ctggcga 7 Hi-PCR-BR catgcggccgccctattattgcaggtattct t 8 95891.doc 18- 200525028

Hi核苷酸序列 GATCCTTT ATG GTT GTT TAC ACT GAC TGC ACT GAA TCC GGT CAG AAC CTG TGC CTG TGC GAA GGC TCT AAC GTT TGC GGC CAG GGC AAC AAA TGC ATC CTG GGC TCT AGA GGC GAA AAA AAT CAA TGC GTT ACT GGC GAA GGT ACT CCG AAA CCG CAG TCT CAC AAC GAC GGC GAC TTT GAA GAA ATC CCG GAA GAA TAC CTG CAA TAA TAGGGC 16 Hi胺基酸序列 Met Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Gin Gly Asn Lys Cys lie Leu Gly Ser Arg Gly Glu Lys Asn Gin Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gin Ser His Asn Asp Gly Asp Phe Glu Glu lie Pro Glu Glu Tyr Leu Gin 17Hi nucleotide sequence GATCCTTT ATG GTT GTT TAC ACT GAC TGC ACT GAA TCC GGT CAG AAC CTG TGC CTG TGC GAA GGC TCT AAC GTT TGC GGC CAG GGC AAC AAA TGC ATC CTG GGC TCT AGA GGC GAA AAA AAT CAA TGC GTT ACT GGC GAA GGT ACT CCG AAA CCG CAG TCT CAC AAC GAC GGC GAC TTT GAA GAA ATC CCG GAA GAA TAC CTG CAA TAA TAGGGC 16 Hi amino acid sequence Met Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Gin Gly Asn Lys Cys lie Leu Gly Ser Arg Gly Glu Lys Asn Gin Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gin Ser His Asn Asp Gly Asp Phe Glu Glu lie Pro Glu Glu Tyr Leu Gin 17

使用DNA片段Hi-AF和Hi-AR做為模板，以及Hi-PCR-AF 和Hi-PCR-AR做為引子，進行PCR以增幅第一個DNA片段 (Hi-A);另使用DNA片段Hi-BF和Hi-BR做為模板，以及 Hi-PCR-BF和Hi-PCR-BR做為引子，進行PCR以增幅第二個 DNA片段（Hi-B)。將前述之模板（各lng)、引子（各0·2μΜ)、 10倍緩衝溶液（10 Ml，其含有 15mM MgCl2、500 mM KC1、 l%Triton X-100、0.1%明膠和 100 mM Tris-HCl，pH7.9)、 dATP、dCTP、dTTP和 dGTP(各 200 μΜ)以及聚合酶（0_5 單位；PromegaCo.，USA)予以混合，達ΙΟΟμΙ之終體積。在進入PCR循環之前，使該反應混合物在94°C下加熱變性5分鐘。反應條件為94°C變性反應45秒、60°C煉合反應45秒以及72°C擴增反應45秒，在此三步驟反應依序總共進行40次循環之後，使該混合物接受72°C 3分鐘之擴增反應’以完成 DNA之延伸。如圖1所示，在H i - A的3 ’ -終端和H i - B的5f -終端設計限制 95891.doc -19- 200525028 酶Zk/的切割位置。利用純化套組（PCR Clean Up-M ; Viogene)純化並回收Hi-A和Hi-B的增幅產物，然後以限制酶刀處理之。以電泳技術分析經酵素處理的DNA片段 Hi-A和Hi-B，並以凝膠萃取套組（Viogene)將DNA從2%瓊脂糖凝膠中回收。將兩個片段連接起來，獲得全長的DNA片段，命名為"Hi”，其含有水蛭素的完整密碼序列。分別分析全長DNA片段（Hi)(SEQ ID NO: 16)和胺基酸序列（SEQ ID NO : 17)，序列内容如表1所示，其中胺基酸34(精胺酸）與 HV1水蛭素的相對應胺基酸（天冬胺酸）不同。前述之酵素切割反應和連接作用（Current Protocols in Molecular Biology， Frederick Μ·Α·等人，2001. John Wiley & Sons，Inc·)係以已知的標準方法進行。實例2 :表現載體的建構 2.1 pBCl-GB-Hir 使用三對引子 Hirlst5，/Hir3，XhoI、Hir2nd57Hir3，XhoI和 Hir3rd5，/Hir3，XhoI，進行三個連續的PCR，將分離自山羊卢-酪蛋白的信號序列加在水蛭素基因（SEQ ID NO : 15)的5’- 終端。該信號序列與引子之核甞酸序列如表2所示。表2 :信號序列的序列及引子信號序列和引子序列(5’-3’）序列編號信號序列 ATGAAGGTCCTCATCCTTGCCTGTCTGGTGGCTCTG GCCATTGCA 9 MKVL 工 LACLVALAIA 10 Hirlst5f 叫 TGGCTCTGGCCATTGCAGTTGTTTACACCGACTG 11 Hir2nd5' TCATCCTTGCCTGTCTGGTGGCTCTGGCCATTGC 12 Hir3rd5f TCGCTCGAGATGAAGGTCCTCATCCTTGCCTGTC 13 Hir3fXhoI TCGCTCGAGTTATTGCAGGTATTCTTCCGGG 14 95891.doc -20- 200525028 將PCR產物連接至PCR2.1載體，產生pCR2.1-GB-Hir。然後以Zh/消化pCR2.1-GB-Hir。將所產生的261個鹼基對的 GB-Hir片段予以純化，並進行選殖***經和鹼性磷酸酶處理的pBCl載體内，產生表現載體PBC1-GB-Hir。圖6顯示pBCl-GB-Hir的質體舆圖。 2.2 pE-aLA-Hi 如圖2所示，分別在Hi-A的3’-終端和Hi-B的5’-終端設計限制酶和的兩個切開位置。以限制酶5am///和 Wi/處理實例1所獲得的編碼水蛭素之全長DNA片段（SEQ ID NO : 16)。以電泳技術分離經酵素處理之DNA片段（215 個鹼基對），並以凝膠萃取套組從2%瓊脂糖凝膠中回收 DNA。將經過純化的DNA片段連接到經處理的載體pEGFP-l(pEGFP-Nl，不含EGFP序列片段），獲得表現載體pE-Hi(3.6kb)。將pE_Hi表現載體轉形至大腸桿菌 NM522勝任細胞内，然後選擇具安匹西林（ampicillin)抗藥性之轉形株。以上的轉形作用係使用已知的方法（Current Protocols in Molecular Biology，Frederick Μ·Α·等人，2001. John Wiley & Sons，Inc·)進行。使用大腸桿菌轉形株增幅 pE-Hi表現載體，並純化該表現載體以進行後續程序。以限制酶心mi//和Z/zo/切開經過純化的表現載體pE-Hi，產生3.6kb之DNA片段，其係進一步以電泳技術分離並從1% 瓊脂糖凝膠中回收。將含有從經處理過之 pceLA-Hfix( S.P· Lin「含有α-乳清蛋白之啟動子和人類血液凝集因子IX之cDNA的雜合基因的建構及表現（Construction 95891.doc -21 - 200525028 and Expression of hybrid gene contained the promoter of α-lactoalbumin and the cDNA of human blood clotting factor IX)」，1996年國立台灣大學畜產學系碩士論文）中回收之a-乳清蛋白的1.9kb DNA片段，連接至前述經酵素處理的 3.6kb DNA片段，以獲得表現載體pE-ceLA-Hi，其可在乳腺細胞中專一地表現水經素。實例3 :轉殖基因的製備 3.1 BCl-GB-Hir 以iVW/和心//限制酶處理由實例2所獲得之pBCl-GB-Hii*表現載體，產生16kb之DNA構築體（BC1_GB-Hir)，其含有山羊酪蛋白啟動子核酸序列、信號核酸序列和前述編碼水蛭素的全長DNA片段（SEQIDNO: 15)。以電泳技術分離16kb之DNA構築體，並從低熔點瓊脂糖凝膠中回收該DNA構築體。以CsCl2分帶技術（banding)和TE 緩衝溶液（10mM Tris-HCl、O.lmM EDTA，ρΗ7·4)透析，予以進一步的純化。以ΤΕ緩衝溶液將經純化的DNA構築體稀釋至1至1 Ong/μΐ的終濃度，並以其做為哺乳動物原核顯微注射的轉殖基因。 3.2 aLA-Hi 以限制酶C/a/和Dra///處理由實例2所獲得之pE-aLA-Hi 表現載體，產生4.77kb之DNA構築體，其含有前述aLA啟動子核酸序列和編碼水蛭素的全長DNA片段（SEQ ID NO : 16)，以及SV40聚腺替酸尾端序列（polyAt ail sequence )。以電泳技術分離4.77kb之DNA構築體，並以凝膠萃取套組 95891.doc -22- 200525028 從1 %瓊脂糖凝膠中回收該DNA構築體，然後以TE緩衝溶液 (10mM Tris-HCl、0.25mM EDTA，pH7.4)將之稀釋至 1至 1 Ong/μΐ的終濃度’以其做為哺乳動物原核顯微注射的轉殖基因。實例4 ··基因轉殖動物的產製才疋供成熟的ICR小鼠或藍瑞斯（Landrace )豬，或其他品系之小鼠或豬，做為產製基因轉殖動物的胚提供者和接受者。 4.1產製小鼠的程序所有的小鼠均飼養在乾淨的實驗室鼠房中，維持在2〇至 26 C，並經由HEPA系統換氣，提供1 〇小時黑暗與丨4小時明冗的期間。播限制地供應新鮮的水和飼料。以腹腔内注射 PMSG使母隻雌性小鼠進行誘發滤泡超數發育，在以注射後48小時之後，以人類絨毛膜***（hCG)注射小鼠誘發***，並在同日與ICR雄性種小鼠交配。翌日將受精卵從輸卵管中沖出，然後以微分干涉對比倒立顯微鏡配合使用Narishige操作系統（manipulator)對原核胚進行顯微操作。將依前述製備之轉殖基因注射到小鼠胚的雄性原核内，並將存活者分成25至30個一組，然後移置到***母動物的輸卵管内。在分娩之後，照顧新生的動物達四欠後切下一小片尾巴，萃取基因體DNA，藉由PCR篩選外源基因（exogene)。 4.2產製基因轉殖豬的程序提供純種的Landrace(L)女豬，至少7個半月大。以i 〇至2 95891.doc -23- 200525028 公斤市售的飼料餵飼動物一天兩次，並無限制地供應水。授乳母豬則餵飼母豬料。在分娩之後28天讓基因轉殖的仔豬離奶。在早上餵食混合Regumate®(其含有0.4%晞丙孕素 (altrenogest) ; 20毫克/天；Intervet，荷蘭）的市售飼料15至 1 8天，使所有的供胚和接受胚之女豬達發情同期化。在最後一次餵食Regumate®之後24小時，以肌肉内注射 PMSG( 1500-2000單位，Intervet，荷蘭），誘發供胚豬之濾泡超數發育。在PMSG注射之後76至78小時，以 hCG(1250-1750單位，Intervet，荷蘭）注射豬隻誘發***，並在hCG注射後24至36小時，以純種L公豬新鮮稀釋的精液，對提供者進行人工授精。在hCG注射後54至56小時，對供胚豬隻進行手術，從輸卵管中以20ml之Dulbeccos’-PBS(購自Gibco/BRL，美國）含 0.4%BSA(購自Fraction V，Sigma，美國）將受精卵沖至培養皿中。在手術之前，使所有豬禁食過夜，並以肌肉内注射 5ml 之賜靜寧（sterinil)(2mg/100kg ， Janssen Pharmaceutical ， Belgium)和 10ml 之硫酸阿托品 (5mg/l 00kg，中國化學製藥，台灣）分別使其鎮靜及抑制唾液分泌，然後在耳靜脈注射治得舒（CitosolR;杏林製藥，曰本）使其麻醉。在整個手術中，藉著吸入福來生 (halothane，含4%氯烷；ICI Ltd.，USA)維持麻醉。以如同對於供胚豬所進行之相同程序，藉手術方式將胚移置其他經發情同期化的***受胚母豬之輸卵管内。 95891.doc -24- 200525028 在室溫下，使用離心機（Hettich EBA 12，德國），以15,〇〇〇至23,500 X g使受精卵離心1 〇至8分鐘以露出原核。以微分干涉對比倒立顯微鏡使用Leica機械操作系統對豬胚進行顯微操作。將轉殖基因注射至受精印之原核或兩細胞期豬胚的核内。在注射25至30個豬胚之後，儘快將胚移置經發情同期化之受胚豬的輸卵管内。鬌 4·3 BCl-GB_Hir之基因轉殖小鼠及豬瓠在BC 1 -GB-Hir轉殖基因的顯微注射之後，將總共275個小鼠胚移至10隻受胚小鼠的輸卵管内。有7隻小鼠懷孕，並生 _ 下29隻仔小鼠。經PCR分析（圖7)和南方墨點雜交作用（圖 8)，證實在它們之中有4隻小鼠（3隻是雄性，丨隻是雌性）是基因轉殖小鼠，將在下文中說明。雖然本發明之BC1 -GB-Hir 構築體長度為16kb，但轉殖基因仍以正常的百分比嵌入小鼠胚胎的基因體内。13.8%(4/29)的成功率指出轉殖基因的長度’對於將外來D N A嵌入胚基因體内，僅引起極少的干擾。此外，只要它們達到性成熟，就讓所有基因轉殖的小鼠籲與一般小鼠交配。大約有48.4%到60.0%的後代從其基因轉殖之親代遺傳到轉殖基因。這樣高的生殖種系傳代率，代表該轉殖基因可穩定嵌入生殖細胞中（表3)。表3 : BCl-GB-Hir轉殖基因的生殖種系傳代率基因轉殖小鼠的親代性別 F1後裔出生F1基因轉殖的數目後裔的數目傳代率 2-1 雄 28 14 50.0 4-4 雌 21 11 52.4 95891.doc -25- 200525028 6_1 6-3 雄雄 25 31 15 15 60.0 48.4 BCl-GB-Hir構築體含有2X/3-乳球蛋白絕緣子元件。這暗示當將/3-乳球蛋白絕緣子元件***哺乳動物之基因體内時，提高了本發明之BC1-GB-Hir構築體的完整***其基因體内並具傳代率之穩定性。另，依前述針對小鼠之基因轉殖方式，以含心酪蛋白啟動子及信號序列之水蛭素轉殖基因，製備基因轉殖豬。總共注射1，259個豬胚，移入48頭受胚豬，其中有丨丨頭懷孕 (22.9/。），共生下66頭仔豬。對仔豬進行篩選，其中有6頭為水蛭素基因轉殖豬（表4)。新生仔豬受胚豬頭數 (%) 胚移置懷孕(％) 出生數 330 14 1(7.14) 5 299 10 3(30.0) 16 308 11 4(36.4) 31 321 13 3(23.1) 14 總計1259 48 4(500)~ 18 表4:水蛭素基因轉殖豬之產制注射 0(0.0) 2(12.5) 1(3.22) 3(21.4) 6(9.1) 根據在本文中描述的方法，進—步產製帶有水蛭素轉殖基因之家畜動物’包括乳羊、乳牛或兔子，以便從其乳汁中收集水經素。 4.4aLA_Hi之基因轉殖小鼠和豬如表5所π，將轉殖基因注射至383個小鼠胚胎和18〇個豬胚月口内，並將胚胎分別移至15和8隻養母動物内。在懷孕之 95891.doc -26- 200525028 其中證實5隻小鼠和後，生出3 0隻新生的小鼠和1 8隻仔豬 1隻仔褚疋基因轉殖動物。素基因轉殖砧车製動物胚數目受胚動物的數目顯微注射胚移置移置懷孕(％) 仔小鼠出生基因轉殖動物Use DNA fragments Hi-AF and Hi-AR as templates, and Hi-PCR-AF and Hi-PCR-AR as primers, and perform PCR to amplify the first DNA fragment (Hi-A); another DNA fragment Hi -BF and Hi-BR are used as templates, and Hi-PCR-BF and Hi-PCR-BR are used as primers, and PCR is performed to amplify the second DNA fragment (Hi-B). The aforementioned template (1 ng each), primers (0.2 μM each), 10-fold buffer solution (10 Ml containing 15 mM MgCl2, 500 mM KC1, 1% Triton X-100, 0.1% gelatin, and 100 mM Tris-HCl , PH 7.9), dATP, dCTP, dTTP and dGTP (200 μM each) and polymerase (0-5 units; PromegaCo., USA) were mixed to a final volume of 100 μl. Prior to entering the PCR cycle, the reaction mixture was heat-denatured at 94 ° C for 5 minutes. The reaction conditions were: 94 ° C denaturation reaction for 45 seconds, 60 ° C blending reaction for 45 seconds, and 72 ° C amplification reaction for 45 seconds. After the three-step reaction was carried out for a total of 40 cycles, the mixture was subjected to 72 ° C. A 3-minute amplification reaction 'to complete the extension of the DNA. As shown in Figure 1, the 3'-terminus of Hi-A and the 5f-terminus of Hi-B design limits 95891.doc -19-200525028 the cleavage position of the enzyme Zk /. The purification products (PCR Clean Up-M; Viogene) were used to purify and recover the amplified products of Hi-A and Hi-B, and then treated them with a restriction enzyme knife. The enzyme-treated DNA fragments Hi-A and Hi-B were analyzed by electrophoresis, and the DNA was recovered from a 2% agarose gel using a gel extraction kit (Viogene). The two fragments were ligated together to obtain a full-length DNA fragment named "Hi", which contains the complete codon sequence of hirudin. The full-length DNA fragment (Hi) (SEQ ID NO: 16) and the amino acid sequence ( (SEQ ID NO: 17), the sequence content is shown in Table 1, wherein the corresponding amino acid (aspartic acid) of amino acid 34 (spermine) and HV1 hirudin is different. The aforementioned enzyme cleavage reaction and connection The effects (Current Protocols in Molecular Biology, Frederick M.A. et al., 2001. John Wiley & Sons, Inc.) were performed using known standard methods. Example 2: Construction of expression vectors 2.1 pBCl-GB-Hir use Three pairs of primers Hirlst5, / Hir3, XhoI, Hir2nd57Hir3, XhoI, and Hir3rd5, / Hir3, XhoI, were subjected to three consecutive PCRs, and the signal sequence isolated from goat lu-casein was added to the hirudin gene (SEQ ID NO: 15 5'-terminal of this signal. The nucleotide sequence of this signal sequence and primer is shown in Table 2. Table 2: Sequence of signal sequence and primer signal sequence and primer sequence (5'-3 ') Sequence number signal sequence ATGAAGGTCCTCATCCTTGCCTGTCTGGTGGCTCTG GCCATT GCA 9 MKVL and LACLVALAIA 10 Hirlst5f are called TGGCTCTGGGGCCATTGCAGTTGTTTACACCGACTG 11 Hir2nd5 'TCATCCTTGCCTGTCTGGTGGCTCTGGCCATTGC 12 Hir3rd5f TCGCTCGAGATGTGGG-CC-CCC-HCC. Digest pCR2.1-GB-Hir. The 261-base-pair GB-Hir fragment generated was purified and cloned into a pBCl vector treated with alkaline phosphatase to produce the expression vector PBC1-GB- Hir. Figure 6 shows the plastid map of pBCl-GB-Hir. 2.2 pE-aLA-Hi As shown in Figure 2, restriction enzymes were designed at the 3'-terminus of Hi-A and the 5'-terminus of Hi-B, respectively. And two incision positions of and. The full length DNA fragment (SEQ ID NO: 16) encoding hirudin obtained in Example 1 was treated with restriction enzymes 5am /// and Wi /. The enzyme-treated DNA fragments (215 base pairs) were separated by electrophoresis and the DNA was recovered from a 2% agarose gel using a gel extraction kit. The purified DNA fragment was ligated to the treated vector pEGFP-1 (pEGFP-Nl, which does not contain an EGFP sequence fragment) to obtain the expression vector pE-Hi (3.6 kb). The pE_Hi expression vector was transformed into competent cells of E. coli NM522, and then an ampicillin-resistant transformed strain was selected. The above transformation is performed using a known method (Current Protocols in Molecular Biology, Frederick M.A. et al., 2001. John Wiley & Sons, Inc.). The E. coli transformant was used to amplify the pE-Hi expression vector, and the expression vector was purified for subsequent procedures. The purified expression vectors pE-Hi were cut with restriction enzymes mi // and Z / zo / to generate a 3.6 kb DNA fragment, which was further separated by electrophoresis and recovered from a 1% agarose gel. Construction and expression of a hybrid gene containing the processed pceLA-Hfix (SP · Lin, "a-whey protein-containing promoter and human hemagglutination factor IX cDNA (Construction 95891.doc -21-200525028 and Expression of hybrid gene contained the promoter of α-lactoalbumin and the cDNA of human blood clotting factor IX "", a 1.9 kb DNA fragment of a-whey protein recovered from the 1996 National Taiwan University Master's Dissertation), ligated to The aforementioned enzyme-treated 3.6 kb DNA fragment to obtain the expression vector pE-ceLA-Hi, which specifically expresses meridin in breast cells. Example 3: Preparation of transgenic genes 3.1 BCl-GB-Hir The pBCl-GB-Hii * expression vector obtained in Example 2 was treated with iVW / and cardio // restriction enzymes to produce a 16 kb DNA construct (BC1_GB-Hir) , Which contains a goat casein promoter nucleic acid sequence, a signal nucleic acid sequence, and the aforementioned full-length DNA fragment encoding hirudin (SEQ ID NO: 15). A 16 kb DNA construct was separated by electrophoresis, and the DNA construct was recovered from a low-melting agarose gel. CsCl2 banding and TE buffer solution (10 mM Tris-HCl, 0.1 mM EDTA, ρM7.4) were dialyzed for further purification. The purified DNA construct was diluted with TE buffer solution to a final concentration of 1 to 1 Ong / μΐ and used as a transgenic gene for mammalian prokaryotic microinjection. 3.2 aLA-Hi treated the pE-aLA-Hi expression vector obtained in Example 2 with restriction enzymes C / a / and Dra /// to generate a 4.77 kb DNA construct containing the aforementioned aLA promoter nucleic acid sequence and encoding the leech Full-length DNA fragment (SEQ ID NO: 16), and SV40 polyAt ail sequence. The DNA construct of 4.77kb was separated by electrophoresis, and the DNA construct was recovered from a 1% agarose gel using a gel extraction kit 95891.doc -22- 200525028, and then a TE buffer solution (10mM Tris-HCl, 0.25 mM EDTA, pH 7.4) was diluted to a final concentration of 1 to 1 Ong / μΐ 'as a transgenic gene for mammalian prokaryotic microinjection. Example 4 · Production system of transgenic animals Only for mature ICR mice or Landrace pigs, or other strains of mice or pigs, as embryo suppliers and recipient. 4.1 Procedures for producing mice All mice are housed in clean laboratory rat rooms, maintained at 20 to 26 C, and ventilated via the HEPA system, providing a period of 10 hours of darkness and 4 hours of lightness . Restricted supply of fresh water and feed. Intraperitoneal injection of PMSG induced female follicles to overgrow, and 48 hours after injection, mice were injected with human chorionic gonadotropin (hCG) to induce ovulation, and on the same day with ICR males Mating. The next day, the fertilized eggs were flushed from the fallopian tubes, and then the differential interference contrast inverted microscope was used in conjunction with the Narishige operating system (manipulator) to perform micromanipulation on the prokaryotes. The transgenes prepared as described above were injected into male pronuclei of mouse embryos, and the survivors were divided into groups of 25 to 30, and then transferred to the fallopian tubes of surrogate mother animals. After childbirth, take care of newborn animals for four owes, cut a small piece of tail, extract genomic DNA, and screen exogenes by PCR. 4.2 Procedures for producing genetically modified pigs Provide pure breed Landrace (L) female pigs, at least 7 1/2 months old. Animals were fed twice daily with i 0 to 2 95891.doc -23- 200525028 kg of commercially available feed and were supplied with unlimited water. Suckling sows are fed sow feed. Transgenic piglets were weaned 28 days after delivery. Commercially available Regumate® (which contains 0.4% altrenogest; 20 mg / day; Intervet, Netherlands) was fed in the morning for 15 to 18 days, bringing all donor and recipient female pigs to Estrus synchronization. Twenty-four hours after the last Regumate® feeding, PMSG (1500-2000 units, Intervet, Netherlands) was injected intramuscularly to induce overgrowth of follicles from donor pigs. 76 to 78 hours after PMSG injection, pigs were injected with hCG (1250-1750 units, Intervet, Netherlands) to induce ovulation, and 24 to 36 hours after hCG injection, freshly diluted semen from purebred L boars were provided to Perform artificial insemination. From 54 to 56 hours after hCG injection, donor pigs were operated on with 20 ml of Dulbeccos'-PBS (purchased from Gibco / BRL, USA) containing 0.4% BSA (purchased from Fraaction V, Sigma, USA) from the fallopian tubes. Wash the fertilized eggs into a petri dish. Prior to surgery, all pigs were fasted overnight and intramuscularly injected with 5 ml of sterinil (2 mg / 100 kg, Janssen Pharmaceutical, Belgium) and 10 ml of atropine sulfate (5 mg / l 00 kg, Sinochem, Taiwan ) Sedated and inhibited saliva secretion, and then anesthetized by injecting Zhishu (CitosolR; Xinglin Pharmaceutical Co., Ltd.) into the ear vein. Throughout the procedure, anesthesia was maintained by inhaling fluoxane (halothane, 4% chloroethane; ICI Ltd., USA). Following the same procedure as for donor pigs, the embryos are surgically transferred into the fallopian tubes of other estrus-synchronized surrogate embryos. 95891.doc -24- 200525028 At room temperature, centrifuge the fertilized eggs at 15,000 to 23,500 X g using a centrifuge (Hettich EBA 12, Germany) for 10 to 8 minutes to expose the pronucleus. Differential intervention contrast microscope was used to differentiate pig embryos using Leica mechanical operating system. The transgenic gene is injected into the nucleus of a fertilized prokaryote or a two-cell stage pig embryo. Immediately after injection of 25 to 30 pig embryos, the embryos are placed in the oviduct of an embryo that has undergone estrus synchronization.鬌 4 · 3 BCl-GB_Hir transgenic mice and pig 瓠 After microinjection of BC 1 -GB-Hir transgenic genes, a total of 275 mouse embryos were transferred to the oviducts of 10 embryonic mice . Seven mice became pregnant and gave birth to 29 pups. PCR analysis (Figure 7) and Southern blot dot hybridization (Figure 8) confirmed that 4 of them (3 were male, only female) were transgenic mice, which will be described below. Although the BC1-GB-Hir construct of the present invention is 16 kb in length, the transgenic gene is still embedded in the gene of the mouse embryo at a normal percentage. A success rate of 13.8% (4/29) indicates that the length of the transgenic gene 'causes only a small amount of interference for embedding foreign DNA into the embryo gene. In addition, as long as they reach sexual maturity, all transgenic mice are called to mate with normal mice. Approximately 48.4% to 60.0% of the offspring inherit from the parent of their gene transfer to the transgene. Such a high reproductive germline passage rate indicates that the transgene can be stably embedded in germ cells (Table 3). Table 3: Reproductive germline passage rates of BCl-GB-Hir transgenic genes. Parental sex of transgenic mice. F1 descendants were born. Number of F1 gene transfers. Number of descendants. 2-1 Male 28 14 50.0 4 -4 Female 21 11 52.4 95891.doc -25- 200525028 6_1 6-3 Male 25 31 15 15 60.0 48.4 The BCl-GB-Hir construct contains 2X / 3-lactoglobulin insulator elements. This implies that when a / 3-lactoglobulin insulator element is inserted into a mammalian gene, the BC1-GB-Hir construct of the present invention is completely inserted into the gene and has a stable passage rate. In addition, according to the aforementioned method for gene transfer in mice, a gene transgenic pig was prepared using a hirudin gene containing a heart casein promoter and a signal sequence. A total of 1,259 pig embryos were injected and 48 embryos were transferred. Among them, 172 were pregnant (22.9 /.), And 66 piglets were born. Piglets were screened and 6 of them were hirudin transgenic pigs (Table 4). Number of newborn piglets with embryos (%) Embryo transfer pregnancy (%) Number of births 330 14 1 (7.14) 5 299 10 3 (30.0) 16 308 11 4 (36.4) 31 321 13 3 (23.1) 14 Total 1259 48 4 (500) ~ 18 Table 4: Production injection of hirudin gene transgenic pigs 0 (0.0) 2 (12.5) 1 (3.22) 3 (21.4) 6 (9.1) According to the method described in this article, further production Production of livestock animals with hirudin transgenic genes includes dairy sheep, cows or rabbits in order to collect hydroquinone from their milk. 4.4 Transgenic mice and pigs with aLA_Hi gene As shown in Table 5, the transgenic genes were injected into the mouths of 383 mouse embryos and 180 pig embryos, and the embryos were transferred to 15 and 8 foster mothers, respectively. In pregnancy 95891.doc -26- 200525028, 5 mice and 5 were confirmed, and 30 newborn mice and 18 piglets were born and 1 piglet gene transgenic animal was born. Gene Transplanting Anvil System Animal Number of Embryos Number of Embryo Microinjected Embryo Translocation Transplantation Pregnancy (%) Puppy Mouse Birth Transgenic Animal

實例5:基因轉殖動物之基因體分析Example 5: Genomic analysis of transgenic animals

在分娩出新生小鼠或小豬之後，在離奶或分娩日，分別採下新生小鼠的尾巴組織或仔豬的耳朵組織，萃取基因體 DNAs做為PCR分析之模版。 5.1 BCl-GB-Hir 利用下列的pBCl正向和逆向引子（表6)，以PCR篩選轉殖基因。表6 :引子之核苷酸序列引子序列(5f-3’）序列編號 pBCl_正向 GATTGACAAGTAATACGCTGTTTCCTC 20 pBCl-逆向 CATCAGAAGTTAAACAGCACAGTTAG 21 將模板DNAs(各l〇〇ng)或BCl-GB-Hir(lng，陽性對照組）加至個別的PCR反應混合物中’並在95 °C下加熱變性反應5 分鐘，然後進入PCR循環。循環反應條件為95°C變性反應 30秒，55°C煉合反應30秒和72°[擴增反應30秒’在總共35 次循環之後，使該混合物經72°C延長2分鐘之擴增反應，以便所有反應產物完成DNA延伸。然後’以電泳技術在2%瓊 95891.doc -27- 200525028 脂糖凝膠中分析PCR產物，觀察到385個鹼基對的DNA條帶。此外，以五ai?/限制酶處理15 /ig之前述基因體DNA，然後藉著電泳在0.8%瓊脂糖凝膠上分離。在鹼性變性作用之後，將基因體DNA轉潰於硝基纖維素膜上，並與P32-標示之水蛭素DNA探針進行雜交，然後以放射自顯術完成顯像。 5.2 aLA-Hi 利用下列特定的引子對以PCR篩選轉殖基因，該等引子對係根據aLA啟動基因序列的上股和水蛭素序列的下股而設計（表7)。表7 :引子核苷酸序列‘ 引子序列(5f-3’）序列編號 PoLA-正向 GCTTCCTAGAACCAACACTACCAG 18 PoLA-逆向 GTCGCCGTCGTTGTGAGACTG 19 將模板DNAs(各100ng)或pE-aLA-Hi(lng，陽性對照組）加至個別的PCR反應混合物中，其含有10-倍PCR緩衝溶液 (10 μ卜包括 15 mM MgCl2、500mM KCM、l%Triton X-100、 0.1% 明膠和 100 mM Tris-HCn，ρΗ7·9)、dATP、dCTP、dTTP 和dGTP(各200 μΜ)、前述引子對（各0·2 μΜ)和0.5單位Tbg 聚合酶。將PCR混合物於94°C加熱變性反應3分鐘，然後進入PCR循環。循環反應條件為94°C變性反應60秒，55°C煉合反應60秒和72°C擴增反應60秒。在總共35次循環之後，使該混合物經歷72°C最後擴增反應3分鐘，以便完成DNA延伸。然後，以電泳技術在2%瓊脂糖凝膠中分析PCR產物， 95891.doc -28- 200525028 觀察到472個鹼基對的DNA條帶（圖5)。實例6 :水蛭素在乳汁中的抗凝集活性及純化以水蛭素抑制凝血酶對色原受質Tos-Gly-Pro-Arg-4_NA 之作用而降低釋放有顏色之4-硝基苯胺（ΝΑ)的能力為基礎，測量在小鼠乳汁中之水蛭素的抗凝集活性（Lyer，L·等人，1995，Thrombosis Res.，78(3) : 259-263)。收集小鼠乳汁，並以14,000 X g離心5分鐘。將上清液與凝血酶溶液 (50U/ml)及 Tris 緩衝溶液（50mMTris，pH8.3，227 mMNaCl) 混合。將不同體積之上清液與凝血酶溶液混合。在37°C下培養10分鐘之後，加入色原受質Tos-Gly-Pro-Arg-4-NA，並在405nm(A4G5)處讀取吸光度1分鐘。使用商業化的天然水蛭素（American Diagnostica Inc·重組的水蛭素#5301，200pg 的小瓶，約2,000ATU)，建立標準曲線。A4G5讀值越低表示抗凝集活性越高。以標準曲線和讀值為基礎，計算乳汁中的水經素之含量。如圖9所示，從本發明之正在泌乳的基因轉殖小鼠中收集到的乳汁，與得自非-基因轉殖小鼠的（正常小鼠乳汁）相比較，具有高程度的抗凝集活性。收集自所有4個基因轉殖系的乳汁表現出範圍從每微升乳汁0· 1到40單位的抗凝集活性，且持續整個泌乳期間（表8)。 95891.doc -29- 200525028 表8 :在基因轉殖小鼠乳汁中之水蛭素的抗凝集活性品系小鼠的數目抗凝集活性(ATU/ml) 第7天第14天第21天 2-l(Fl) 14 12,000-40,000 3,000·5,400 3,600-4,600 4_4(F0) 1 350 340 480 6-l(Fl) 15 100-450 110-151〇〇 380-1,200 6-3(Fl) 15 350-3,200 2,900-3,600 2,200-3,200 F 0 :基因轉殖的親代 F1 :基因轉殖後裔的第1代如前所述，總共成功產生4個水蛭素基因轉殖小鼠品系，其乳汁萃取物具有高程度的抗凝集活性。以上結果證實本發明之BCl-GB-Hir構築體可在基因轉殖動物的乳腺中，有效地表現高程度的水蛭素。此外，以高程度表現的期間持續至少三週，尤其是2-1基因轉殖品系，與先前技藝相比較，有助於產生大量的水蛭素。根據本發明，使用該啟動基因有利於產生大量的重組蛋白質。收集本發明之水蛭素基因轉殖小鼠品系之乳汁，以葡聚糖凝膠進行分子篩管柱層析後，再進一步以RP-HPLC純化。結果如圖10所示。實例7 :在轉形的乳腺細胞株中藉pE-aLA-Hi表現水蛭素 7.1培養乳腺細胞株在37°C，5%C02下，將購自食品工業發展研究所（台灣，新竹）之細胞銀行的小鼠乳腺上皮細胞株 NMuMG(BCRC60087)培養在含有4.5mg/ml之葡萄糖和10% 95891.doc -30- 200525028 胎牛血清（FBS)的 Dulbecco Modified Eagles Medium(DMEM)培養基中。生長在以上培養條件中的 NMuMG細胞顯露出單一多邊形的外觀，沒有觸角，且不具有分化乳腺細胞的功能，例如，運送或分泌蛋白質到細胞外。然而，若在培養基中加入5/xg/ml之胰島素、5/xg/ml之催乳激素和1 Mg/ml之***（dexamethasome)，並將一層基質凝膠（Matrixgel)(50pl/cm2，購自 BD BioSciences， Clontech，USA)塗覆在培養皿的底部，在培養24小時之後，可觀察到NMuMG細胞的清楚細胞群落。每個群落包括數萬個聚集的細胞，並形成中空半球形的形態，其類似在活體内乳腺之泌乳細胞群的外觀。 7.2乳腺細胞株的轉形將實例2所述之pE-ceLA-Hi表現載體（5/ig，50μ1)均勻地與微脂粒（ΙΟΟμΙ，SuperFect，QIAGENE)混合，然後在該混合物中加入不含血清的DMEM培養基（850μ1)，以產生"DNA-微脂粒-培養基溶液’’。在不添加荷爾蒙（胰島素、催乳激素和***）的基質凝膠上，於六孔培養皿中培養NMuMG 細胞至60至80%的密度，以磷酸-緩衝之生理鹽水（PBS， ρΗ7·4)沖洗3次。將"DNA-微脂粒-培養基溶液”加至細胞中，然後在37°C，5%C02下培養1小時，並在其中加入2ml 含有20%FBS的DMEM培養基。在連續培養細胞24小時之後，移出含有DNA和微脂粒的溶液，並加入含有10% FBS 的DMEM培養基。亦加入抗生素Geneticin(500 /xg/ml， G41 8，Sigma)，以用於選擇經轉形的細胞，且隨後不規律 95891.doc •31 - 200525028 地置換培養液。在兩次連續世代之後，獲得在G41 8的存在下，穩定具有pE_aLA-Hi表現載體的NMuMG細胞 (pE‘A-Hi/NMuMG)。實例8 :在分離的乳腺組織表現水蛭素 8.1分離乳腺組織在分娩後第11天犧牲泌乳的ICR雌鼠，並分離其乳腺組織。以1倍PBS沖洗在活體外的乳腺組織3次，並以1，000rpm 之低速離心15分鐘，以便洗掉乳汁。將乳腺組織切成8立方毫米的碎片。將0.25克組織的碎片懸浮於0.8ml的DMEM培養基中。 8.2乳腺組織的轉形作用藉著電穿透技術完成乳腺組織的轉形作用。將pE-aLA-Hi 質體DNA(40/ig)加至上述乳腺組織中。在均勻地混合乳腺組織與DNA達10分鐘之後，將其倒入寬0.4公分的電穿透小管中，並在200伏特/公分，50毫秒的條件下，以電穿透器（ECM 2001，BTX，USA)處理6次。將經過處理的乳腺組織移至35毫米的培養内，並將培養基換成含有5/xg/ml之胰島素、5 jitg/ml之催乳激素、1 jug/ml之***和10%FBS 的DMEM培養基。在37°C，5%C02的恆溫箱中培養經過轉形的乳腺組織達 48小時，然後均質化，獲得均質的組織溶液。依前述方式測定在經轉形之乳腺組織的均質萃取物中之水蛭素的抗凝集活性。 8.3分析在經轉形之乳腺組織中之水蛭素的生物活性 95891.doc -32- 200525028 以各種含量（0_03、0.06、0· 13、0.25、0.5、1和 2 mg總蛋白質）之經轉化的乳腺組織之均質萃取物，以及各種含量的培養基（0.15、0.31、0.62、1.25、2.5、5和 10 μΐ)，依前述方式測定抗凝集之生物活性。實例9:分析經轉形之乳腺細胞株所表現之水蛭素的生物活性 9.1以乳腺細胞株表現水蛭素使實例7所獲得的pE-aLA-Hi轉形乳腺細胞株 (pE-aLA-Hi/NMuMG)，在 37°C，5%C02下，在塗覆基質凝膠的培養皿中，培養於含有荷爾蒙（胰島素、催乳激素和地塞米松）及500 /xg/ml之抗生素geneticin的乳腺細胞培養基中。當細胞聚落形成類似乳腺濾泡的中空半球時，將細胞培養物移至乾淨的試管中。以0.25%胰蛋白酶溶液處理使細胞自培養皿中分離。以1，000 rpm離心5分鐘而收集細胞。將細胞再懸浮於1 ml之高張溶液（25%蔗糖、lml EDTA和 Tris-HCl，ρΗ7·5)中，並在室溫下培養15分鐘。然後，利用起音波震盛器將細胞打破，並以6,000 rpm離心10分鐘，以移除細胞片段。所獲得的上清液是含有水經素之乳腺細胞的均質萃取物。 9 · 2抗凝集生物活性的判定對前述乳腺細胞的均質萃取物和細胞培養液，分別進行水蛭素之抗凝集生物活性的判定。測定在乳腺細胞之均質萃取物中的蛋白質總量，做為在抗凝集生物活性分析中加入均質萃取物的基礎和單位。提供於分析緩衝溶液（〇12M NaCl、0.01M磷酸鈉、 95891.doc -33 - 200525028 0.01。/以&^和0.1%牛血清白蛋白，？117.4)中濃度為0.2?111〇4 的牛凝血酶。分別將各種濃度（0.04、0_08、0.16、0.32、0·64、 1 ·28和2.5 6pmole)的市售天然水經素、各種含量（0·03、 0.06、0.13、0.25、0·5、1和2gg之總蛋白質）的前述乳腺細胞之均質萃取物，以及各種含量（0.15、0.31、0_62、1.25、 2.5、5和1〇μ1)的培養液，與前述牛凝血酶（50 μΐ)混合。在 24°C下培養1分鐘之後，加入1〇〇μ1經分析緩衝溶液1〇倍稀釋的人類血清，並充份混合20秒，待反應15分鐘之後，測定該反應混合物在A4G5處的吸光度。若A4C5讀值越低，則意指抗凝集程度越高。如圖3和4中所示，當天然水蛭素的濃度為〇.16prn〇le時，幾乎沒有凝集，而在乳腺細胞之均質萃取物和培養液的量分別為0.25 mg和10 μ1時，會發生類似的無凝集狀態。當a4〇5 頃值為0.05之抗凝集反應所需的天然水蛭素、乳腺細胞之均質萃取物和培養液的濃度分別為0.52pmole、0·056 mg和〇·95 μ卜【圖式簡單說明】圖1顯示編碼本發明之水經素之聚核苷酸片段之合成流程。圖2顯示本發明之pE-ceLA-Hi的建構。圖3為顯示天然水蛭素以及本發明之經轉形的乳腺細胞和組織之均質萃取物的血漿凝集圖。圖4為顯示天然水蛭素以及本發明之經轉形的乳腺細胞和組織之培養基的血漿凝集圖。 95891.doc -34- 200525028 圖5顯示本發明之aLA_水蛭素基因轉殖小鼠（a)和豬⑺）的PCR分析。符號” + "、"_，，、”Tg”和”w”分別代表陽性對照組、陰性對照組、基因轉殖動物和水。圖6顯示本發明之pBC1_GB-Hir表現載體的質體興圖。圖7顯示根據本發明之基因轉殖（Tg)小鼠的pcR分析。M 道代表lkb標記；i至n道分別代表不同的小鼠品系 2-l(Tg)、2-2、2-3、2·4、4]、4-2、4-3、4-4(Tg)、6-l(Tg)、 6-2和6-3(Tg) ;，，+ ”道代表陽性對照組；，，_，，道代表陰性對照組；以及，’H20"道代表以水做為模板。圖8顯示根據本發明之基因轉殖小鼠的南方墨點分析。m 道代表XDNA/7//M///標記；以及道分別代表基因轉殖小鼠品系2-1、4-4、6·1、6-3、NC(這是正常的對照組，即非-基因轉殖小鼠基因體）、一個副本（即正常對照組小鼠的基因體與一個副本之轉殖基因混合），以及1〇個副本（即正常對照組小鼠的基因體與10個副本之轉殖基因混合）。圖9(A)為顯示天然水蛭素之抗凝集活性的標準曲線圖。圖9(B)為顯示根據本發明之基因轉殖小鼠的乳汁中的水虫至素之抗凝集活性圖。曲線，，--，，代表做為陰性對照組之正常小鼠乳汁的抗凝集活性。曲線”-□·，，代表根據本發明之基因轉殖小鼠乳汁的抗凝集活性。曲線”-△_，，代表做為陽性對照組的含有不同含量之天然水蛭素（第1點·· 5〇ng，第2點·· l〇〇ng，第3點：200ng)的正常小鼠乳汁的抗凝集活性。圖10A係顯示以Sephadexg-G50管柱純化基因轉殖小鼠之乳汁所含水蛭素之結果；以及圖10B係顯示再進一步使用 RP-HPLC之純化結果。 95891.doc -35-After giving birth to newborn mice or piglets, on the day of weaning or childbirth, the tail tissues of newborn mice or ear tissues of piglets were collected, respectively, and genomic DNAs were extracted as templates for PCR analysis. 5.1 BCl-GB-Hir The following pBCl forward and reverse primers (Table 6) were used to screen for transgenic genes by PCR. Table 6: Nucleotide sequence of primers Primer sequence (5f-3 ') Sequence number pBCl_forward GATTGACAAGTAATACGCTGTTTCCTC 20 pBCl-reverse CATCAGAAGTTAAACAGCACAGTGT 21 template DNAs (100ng each) or BCl-GB-Hir (lng, positive) Control group) Add to individual PCR reaction mixtures and heat denaturation reaction at 95 ° C for 5 minutes before entering the PCR cycle. Cycling reaction conditions were denaturing reaction at 95 ° C for 30 seconds, mixing reaction at 55 ° C for 30 seconds, and 72 ° [30 second amplification reaction 'After a total of 35 cycles, the mixture was amplified by 72 ° C for 2 minutes. Reaction so that all reaction products complete DNA extension. Then, the PCR product was analyzed by electrophoresis on a 2% agar 95891.doc -27- 200525028 liposugar gel, and a DNA band of 385 base pairs was observed. In addition, 15 / ig of the aforementioned genomic DNA was treated with five ai / restriction enzymes, and then separated on a 0.8% agarose gel by electrophoresis. After alkaline denaturation, the genomic DNA was transfected on a nitrocellulose membrane, and hybridized with a P32-labeled hirudin DNA probe, and then imaging was performed by autoradiography. 5.2 aLA-Hi The following specific primer pairs were used to screen transgenic genes by PCR. These primer pairs were designed based on the upper strand of the aLA promoter gene sequence and the lower strand of the hirudin sequence (Table 7). Table 7: Primer nucleotide sequences 'Primer sequence (5f-3') Sequence number PoLA-forward GCTTCCTAGAACCAACACTACCAG 18 PoLA-reverse GTCGCCGTCGTTGTGAGACTG 19 Template DNAs (100ng each) or pE-aLA-Hi (lng, positive control group) Add to individual PCR reaction mixtures containing 10-fold PCR buffer solution (10 μB including 15 mM MgCl2, 500 mM KCM, 1% Triton X-100, 0.1% gelatin, and 100 mM Tris-HCn, ρΗ7.9 ·) DATP, dCTP, dTTP, and dGTP (200 μM each), the aforementioned primer pair (0.2 μM each), and 0.5 unit Tbg polymerase. The PCR mixture was denatured by heating at 94 ° C for 3 minutes, and then entered into the PCR cycle. The cyclic reaction conditions were denaturing reaction at 94 ° C for 60 seconds, refining reaction at 55 ° C for 60 seconds, and amplification reaction at 72 ° C for 60 seconds. After a total of 35 cycles, the mixture was subjected to a final amplification reaction at 72 ° C for 3 minutes to complete the DNA extension. Then, the PCR products were analyzed on a 2% agarose gel by electrophoresis, and a band of 472 base pairs of DNA was observed at 95891.doc -28-200525028 (Figure 5). Example 6: Anticoagulant activity and purification of hirudin in milk. Hirudin inhibits the action of thrombin on the chromogen receptor Tos-Gly-Pro-Arg-4_NA to reduce the release of colored 4-nitroaniline (NA). ) Based on the ability to measure the anti-agglutination activity of hirudin in mouse milk (Lyer, L. et al., 1995, Thrombosis Res., 78 (3): 259-263). Mouse milk was collected and centrifuged at 14,000 X g for 5 minutes. The supernatant was mixed with a thrombin solution (50 U / ml) and a Tris buffer solution (50 mM Tris, pH 8.3, 227 mM NaCl). Mix different volumes of supernatant with thrombin solution. After incubation at 37 ° C for 10 minutes, the chromogen receptor Tos-Gly-Pro-Arg-4-NA was added, and the absorbance was read at 405 nm (A4G5) for 1 minute. A commercially available natural hirudin (American Diagnostica Inc. recombinant hirudin # 5301, 200 pg vial, approximately 2,000 ATU) was used to establish a standard curve. A lower A4G5 reading indicates higher anti-agglutination activity. Based on the standard curve and readings, calculate the amount of hydrolysate in milk. As shown in Figure 9, milk collected from lactating transgenic mice according to the present invention has a higher degree of anticoagulation compared to non-gene transgenic mice (normal mouse milk). Set active. Milk collected from all four transgenic lines showed anti-agglutination activity ranging from 0.1 to 40 units per microliter of milk and continued throughout the lactation period (Table 8). 95891.doc -29- 200525028 Table 8: Number of anti-agglutination activity of hirudin in the milk of transgenic mice Anti-agglutination activity (ATU / ml) Day 7 14 Day 21 -l (Fl) 14 12,000-40,000 3,000 · 5,400 3,600-4,600 4_4 (F0) 1 350 340 480 6-l (Fl) 15 100-450 110-151 00380-1,200 6-3 (Fl) 15 350- 3,200 2,900-3,600 2,200-3,200 F 0: genetically transgenic parent F1: the first generation of genetically transgenic descendants As mentioned previously, a total of 4 hirudin gene transgenic mouse lines were successfully produced, the milk extract of which has High degree of anti-agglutination activity. The above results confirm that the BCl-GB-Hir construct of the present invention can effectively express hirudin in the mammary glands of transgenic animals. In addition, periods of high performance lasting at least three weeks, especially 2-1 transgenic lines, helped to produce large amounts of hirudin compared to previous techniques. According to the present invention, the use of this promoter gene facilitates the production of a large amount of recombinant protein. The milk of the hirudin gene transgenic mouse strain of the present invention was collected, and subjected to molecular sieve column chromatography using a dextran gel, and further purified by RP-HPLC. The results are shown in Figure 10. Example 7: Using pE-aLA-Hi in transformed breast cell lines to express hirudin7.1 cultured breast cell lines at 37 ° C, 5% C02, will be purchased from cells of the Food Industry Development Institute (Hsinchu, Taiwan) The bank's mouse mammary epithelial cell line NmuMG (BCRC60087) was cultured in Dulbecco Modified Eagles Medium (DMEM) medium containing 4.5 mg / ml glucose and 10% 95891.doc -30- 200525028 fetal bovine serum (FBS). The NMuMG cells grown in the above culture conditions show a single polygonal appearance, have no antennae, and do not have the function of differentiating breast cells, for example, transporting or secreting proteins to the outside of the cell. However, if 5 / xg / ml of insulin, 5 / xg / ml of prolactin and 1 Mg / ml of dexamethasome are added to the medium, and a layer of Matrixgel (50 pl / cm2, (Built from BD BioSciences, Clontech, USA) was coated on the bottom of the petri dish. After 24 hours of culture, a clear cell population of NmuMG cells was observed. Each colony contains tens of thousands of aggregated cells and forms a hollow hemispherical morphology that resembles the appearance of a group of lactating cells in the breast in a living body. 7.2 Transformation of Mammary Gland Cell Lines The pE-ceLA-Hi expression vector (5 / ig, 50 μ1) described in Example 2 was uniformly mixed with the liposomes (100 μI, SuperFect, QIAGENE). Serum in DMEM medium (850 μl) to produce " DNA-Liposome-Media Solution ". NmuMG cells were cultured in a six-well Petri dish on a matrix gel without added hormones (insulin, prolactin and dexamethasone) to a density of 60 to 80% in phosphate-buffered saline (PBS, ρΗ7.4 · 4). ) Rinse 3 times. "DNA-liposome-medium solution" was added to the cells, and then cultured at 37 ° C, 5% C02 for 1 hour, and 2 ml of DMEM medium containing 20% FBS was added thereto. The cells were continuously cultured for 24 hours After that, the solution containing DNA and liposomes was removed, and DMEM medium containing 10% FBS was added. The antibiotic Geneticin (500 / xg / ml, G41 8, Sigma) was also added for selection of transformed cells, and Subsequently, the culture medium was replaced irregularly at 95891.doc • 31-200525028. After two consecutive generations, NmuMG cells (pE'A-Hi / NMuMG) with stable pE_aLA-Hi expression vector were obtained in the presence of G41 8. Example 8: The expression of hirudin in the isolated breast tissue8.1 Isolate the breast tissue ICR female rats sacrificing lactation on the 11th day after delivery and isolate their breast tissue. The breast tissue in vitro was washed 3 times with 1x PBS, and Centrifuge at 1,000 rpm for 15 minutes at low speed to wash off the milk. Cut breast tissue into 8 cubic millimeter pieces. Suspend 0.25 g of tissue fragments in 0.8 ml of DMEM medium. 8.2 Transformation of breast tissue by electricity Penetration technology completes the breast Tissue transformation effect. PE-aLA-Hi plastid DNA (40 / ig) was added to the above breast tissue. After the breast tissue and DNA were mixed uniformly for 10 minutes, it was poured into a 0.4 cm wide electro-passage Through a small tube and under 200 volts / cm, 50 milliseconds, treated with an electric penetrator (ECM 2001, BTX, USA) 6 times. The treated breast tissue was transferred to a 35 mm culture, and The medium was changed to DMEM containing 5 / xg / ml of insulin, 5 jitg / ml of prolactin, 1 jug / ml of dexamethasone, and 10% FBS. The culture was carried out in a 37 ° C, 5% CO2 incubator. The transformed breast tissue is up to 48 hours, and then homogenized to obtain a homogeneous tissue solution. The anticoagulant activity of hirudin in the homogeneous extract of the transformed breast tissue is determined as described above. 8.3 Analysis of the transformed breast tissue Biological activity of hirudin in breast tissue 95891.doc -32- 200525028 Homogeneous extracts of transformed breast tissue in various contents (0_03, 0.06, 0.13, 0.25, 0.5, 1 and 2 mg total protein) , And various levels of media (0.15, 0.31, 0.62, 1.25, 2.5, 5 And 10 μΐ), the biological activity of anti-agglutination was measured in the manner described above. Example 9: Analysis of the biological activity of hirudin expressed by the transformed breast cell line 9.1 The pE obtained in Example 7 was expressed as hirudin by the breast cell line -aLA-Hi transformed breast cell line (pE-aLA-Hi / NMuMG), cultured in a matrix gel-coated petri dish at 37 ° C, 5% C02, and cultured in hormone-containing (insulin, prolactin, and Dexamethasone) and 500 / xg / ml antibiotic geneticin in breast cell culture medium. When the cell colony forms a hollow hemisphere that resembles a breast follicle, transfer the cell culture to a clean test tube. Cells were separated from the dish by treatment with a 0.25% trypsin solution. Cells were collected by centrifugation at 1,000 rpm for 5 minutes. The cells were resuspended in 1 ml of hypertonic solution (25% sucrose, 1 ml of EDTA and Tris-HCl, pH 7.5) and incubated at room temperature for 15 minutes. The cells were then broken using an acoustic shock jar and centrifuged at 6,000 rpm for 10 minutes to remove cell fragments. The resulting supernatant was a homogeneous extract of breast cells containing hydrocele. 9.2 Determination of anti-agglutination biological activity For the aforementioned homogeneous extract of breast cells and cell culture fluid, the anti-agglutination biological activity of hirudin was determined respectively. The total amount of protein in the homogeneous extract of breast cells was determined as the basis and unit for adding the homogeneous extract in the anti-agglutination biological activity analysis. Provided in analysis buffer solution (0.12M NaCl, 0.01M sodium phosphate, 95891.doc -33-200525028 0.01. / With 0.1% and 0.1% bovine serum albumin,? 117.4) bovine at a concentration of 0.2 to 11104 Thrombin. Commercially available natural hydrochloride at various concentrations (0.04, 0_08, 0.16, 0.32, 0.64, 1.28, and 2.5 6pmole), and various contents (0.03, 0.06, 0.13, 0.25, 0.5, 1 And 2gg total protein) of the aforementioned homogeneous extracts of breast cells, and various contents (0.15, 0.31, 0-62, 1.25, 2.5, 5 and 10 μ1) of the culture medium were mixed with the aforementioned bovine thrombin (50 μΐ). After 1 minute incubation at 24 ° C, 100 μl of human serum diluted 10 times in the analysis buffer solution was added and mixed thoroughly for 20 seconds. After 15 minutes of reaction, the absorbance of the reaction mixture at A4G5 was measured. A lower A4C5 reading means a higher degree of anti-agglutination. As shown in Figures 3 and 4, when the concentration of natural hirudin is 0.16 prnole, there is almost no agglutination, and when the amount of homogenous extract and culture fluid of breast cells is 0.25 mg and 10 μ1, respectively, A similar non-agglutination state occurred. The concentration of natural hirudin, homogenous extracts of breast cells and culture medium required for anti-agglutination reaction with an a value of 0.05 at 0.05 were 0.52 pmole, 0.556 mg and 0.95 μb, respectively [simple diagram [Description] FIG. 1 shows a synthetic flow of a polynucleotide fragment encoding a hydrochloride of the present invention. Figure 2 shows the construction of pE-ceLA-Hi of the present invention. Figure 3 is a plasma agglutination diagram showing natural hirudin and a homogeneous extract of transformed breast cells and tissues of the present invention. Figure 4 is a plasma agglutination diagram showing natural hirudin and the culture medium of transformed breast cells and tissues of the present invention. 95891.doc -34- 200525028 Fig. 5 shows a PCR analysis of aLA_hirudin gene transgenic mice (a) and porcine pupae) according to the present invention. The symbols "+", ",", ", Tg", and "w" represent the positive control group, the negative control group, the transgenic animal, and water, respectively. Figure 6 shows the plastid of the pBC1_GB-Hir expression vector of the present invention. Figure 7 shows the pcR analysis of transgenic (Tg) mice according to the present invention. Lanes M represent the lkb markers; lanes i to n represent the different mouse strains 2-l (Tg), 2-2, respectively. 2-3, 2 · 4, 4], 4-2, 4-3, 4-4 (Tg), 6-l (Tg), 6-2, and 6-3 (Tg); ,, + " The positive control group;, _ ,, Tao represents the negative control group; and, 'H20 " Tao represents using water as a template. Figure 8 shows Southern blot analysis of genetically transgenic mice according to the present invention. m lanes represent XDNA / 7 // M /// markers; and lanes represent transgenic mouse strains 2-1, 4-4, 6.1, 6-3, and NC (this is a normal control group, ie Non-gene transgenic mouse genomes), one copy (ie, the normal control mice's genome is mixed with one copy of the transgenic genes), and 10 copies (the normal control mice's genome and 10 Copies of the transgenic genes). FIG. 9 (A) is a standard curve diagram showing the anticoagulant activity of natural hirudin. Fig. 9 (B) is a graph showing the anti-agglutination activity of water insects in the milk of a transgenic mouse according to the present invention. The curve,-,, represents the anticoagulant activity of normal mouse milk as a negative control group. The curve "-□ ·" represents the anticoagulant activity of the milk of the transgenic mice according to the present invention. The curve "-△ _" represents the positive control group containing different levels of natural hirudin (point 1 · · 50 ng, point 2 · 100 ng, point 3: 200 ng) The anticoagulant activity of normal mouse milk. Fig. 10A shows the results of purifying the hydradin in the milk of transgenic mice with Sephadexg-G50 column; and Fig. 10B shows the results of further purification using RP-HPLC. 95891.doc -35-

Claims

200525028 十、申請專利範圍： 1. 2. -種核酸構築’其包括以可操作之方式結合的酷蛋白基因啟動子、信號序列和編碼水坦素的聚核誓酸片段。根據請求項1之核酸構築，其中該路蛋白基因啟動子係分離自山羊/?-酪蛋白基因。 3. 5. 根據請求項1之核酸構築 NO: 15或16之核：y：酸序根據請求項1之核酸構 NO ·· 9之核甞酸序列。根據請求項1之核酸構築蛋白絕緣子。 ’其中該聚核菩酸片段具SEQ ID 列。築’其中該信號序列具SEQ ID ’其進一步包括一或多個/3-乳球200525028 X. Scope of patent application: 1. 2.-A nucleic acid construct 'which includes a cool protein gene promoter, a signal sequence, and a polynuclear acid fragment encoding silytanin that are operably combined. The nucleic acid construct according to claim 1, wherein the road protein gene promoter is isolated from a goat /?-Casein gene. 3. 5. Nucleic acid construction according to claim 1 NO: 15 or 16 core: y: acid sequence Nucleic acid sequence according to claim 1 nucleic acid construction NO ·· 9. A protein insulator constructed from the nucleic acid of claim 1. 'Wherein the polynuclear acid fragment has a column of SEQ ID. Building ’wherein the signal sequence has SEQ ID’ which further includes one or more / 3-lactoglobulins

6· —種產製水蛭素之方法，求項1至5項中任一項之核從該哺乳動物收集其體液回收水經素。其步驟包括製得基因體含有請酸構築之基因轉殖哺乳動物；、分泌液或產物；以及從其中 7.根據請求項6之方法，其中該哺乳動物係緒、牛、山羊、6. · A method for producing hirudin, which seeks the nucleus of any one of items 1 to 5 and collects bodily fluids from the mammal and recovers hydrocele. The steps include preparing a transgenic mammal whose gene body contains an acid construct; a secretion or product; and a method therefrom according to claim 6, wherein the mammal is a mammal, a cow, a goat,

綿羊、馬、駱駝或嚙齒動物。康月长員6之方法，其係自該嗔乳動物收集乳汁、血液、尿液、及***，以及從其中回收水虫至素。 9根據.月求項8之方法，其係自該哺乳動物收集乳汁以及從其中回收水蛭素。 1〇·、種產製水蛭素之方法，其步驟包括提供基因體含有請求項1至5中任—項之核酸構築之基因轉殖哺乳動物·從 X 土因轉殖哺乳動物分離組織或細胞·在適合表現水虫至 95891.doc 200525028 素之條件下培養該細胞或組織；以及從該組織或細胞回收水經素。 11. 12. 13. 14. 15. 16. 17. 根據請求項1 〇之方法，其中自該哺乳動物分離之組織或細胞為乳腺組織或乳腺細胞。種表現載體，其包括請求項1至5中任一項之核酸構築及複製起點。一種經轉形的哺乳動物細胞或組織，其包括根據請求項 12項之表現載體。根據凊求項13之細胞或組織，其係小鼠乳腺上皮細胞株。根據請求項13之細胞或組織，可係衍生自非人類之豬、牛山年綿羊、馬、路駆或ϋ齒齒動物。一種產製水經素之方法，其步驟包括提供請求項13項之經轉形的哺乳動物細胞或組織；在適合表現水蛭素之條件下培養該細胞或組織，以及從該細胞或組織回收水蛭素。根據請求項16之方法，其中該哺乳動物細胞或組織係乳腺細胞或乳腺組織。 95891.docSheep, horse, camel or rodent. The method of Kangyue senior 6 is to collect milk, blood, urine, and semen from the suckling animal, and recover water insects from it. 9 The method of claim 8 according to the month, which involves collecting milk from the mammal and recovering hirudin therefrom. 10. A method for producing hirudin, comprising the steps of providing a gene transgenic mammal whose gene body contains the nucleic acid construct of any one of the items 1 to 5 and isolating a tissue or cell from the X soil transgenic mammal -Culturing the cell or tissue under conditions suitable for the expression of water insects to 95891.doc 200525028; and recovering hydrometeor from the tissue or cell. 11. 12. 13. 14. 15. 16. 17. The method according to claim 10, wherein the tissue or cell isolated from the mammal is a breast tissue or a breast cell. A performance vector comprising the nucleic acid construction and replication origin of any one of claims 1 to 5. A transformed mammalian cell or tissue comprising a performance vector according to claim 12. The cell or tissue according to claim 13, which is a mouse mammary epithelial cell line. The cells or tissues according to claim 13 may be derived from non-human pigs, cattle, sheep, horses, coots, or fangs. A method for producing hydroquinone, comprising the steps of providing transformed mammalian cells or tissues according to claim 13; culturing the cells or tissues under conditions suitable for the expression of hirudin, and recovering hirudin from the cells or tissues Vegetarian. The method according to claim 16, wherein the mammalian cell or tissue is a breast cell or a breast tissue. 95891.doc