JPH0751065A - Glycoprotein 39 gene - Google Patents
Glycoprotein 39 geneInfo
- Publication number
- JPH0751065A JPH0751065A JP3508592A JP3508592A JPH0751065A JP H0751065 A JPH0751065 A JP H0751065A JP 3508592 A JP3508592 A JP 3508592A JP 3508592 A JP3508592 A JP 3508592A JP H0751065 A JPH0751065 A JP H0751065A
- Authority
- JP
- Japan
- Prior art keywords
- glycoprotein
- gene
- cdna
- ser
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は新規なムチン糖タンパク
質39遺伝子に関し、更に詳細には腫瘍マーカー、免疫
異常マーカーあるいは各種炎症性疾患マーカーとして有
用なヒト由来のムチン糖タンパク質39のコアタンパク
質をコードする塩基配列を含有する遺伝子に関する。TECHNICAL FIELD The present invention relates to a novel mucin glycoprotein 39 gene, and more specifically, codes for a human-derived mucin glycoprotein 39 core protein useful as a tumor marker, an immune abnormality marker or various inflammatory disease markers. The present invention relates to a gene containing a nucleotide sequence that
【0002】[0002]
【従来の技術】一般に癌化した細胞の細胞膜には正常な
細胞とは異なる糖タンパク質や糖脂質などの複合糖質が
存在することが知られている。またガンを診断するに際
し、癌患者において特異的に産生されるタンパク抗原や
糖鎖抗原を測定する方法が行なわれている。その例とし
ては、癌胎児抗原(CEA) 、α−フェトプロテイン、CA19
−9などの測定による消化器系癌の診断等が知られてい
る〔村松喬,日本臨床,44, p.337-344(1986) ;神奈木
玲児,臨床病理,35, p.1247-1264(1986) ;医学のあゆ
み,106巻,5号,第5土曜特集,235〜250頁(1978
年)〕。2. Description of the Related Art Generally, it is known that glycoproteins such as glycoproteins and glycolipids different from those of normal cells are present in the cell membrane of cancerous cells. Further, in diagnosing cancer, a method of measuring a protein antigen or a sugar chain antigen specifically produced in a cancer patient is performed. Examples include carcinoembryonic antigen (CEA), α-fetoprotein, CA19
Diagnosis of digestive system cancer by measurement of -9 etc. is known [Takashi Muramatsu, Japan Clinic, 44 , p.337-344 (1986); Reiji Kanaki, Clinicopathology, 35 , p.1247-1264. (1986); History of Medicine, Vol. 106, No. 5, 5th Saturday, 235-250 (1978)
Year)〕.
【0003】[0003]
【発明が解決しようとする課題】しかしながら、従来の
各種癌抗原測定を利用するガンの診断法は適用できる癌
の種類が比較的限られていたり、健常人や肝炎等の他疾
患との交差反応がおこるなどの問題点があり、より広範
な種類の癌に適用できる診断法又は特異性の高い診断法
が望まれている。また、種々の免疫異常応答に基づく疾
病や各種炎症性疾患においても適確な診断法が望まれて
いる。そして、かかる疾患の診断に利用出来る腫瘍マー
カー、免疫異常マーカーあるいは各種炎症性疾患マーカ
ーとなり得る新たな糖タンパク質及びそのコアタンパク
質をコードする遺伝子の開発が切望されている。However, the conventional cancer diagnostic methods using various types of cancer antigen measurement are relatively limited in the types of cancer that can be applied, and cross-react with other diseases such as healthy people and hepatitis. Therefore, a diagnostic method applicable to a wider range of cancers or a highly specific diagnostic method is desired. Further, an accurate diagnostic method is desired for diseases caused by various immune abnormal responses and various inflammatory diseases. Then, the development of new glycoproteins and genes encoding their core proteins, which can be used as tumor markers, immunological abnormality markers or various inflammatory disease markers that can be used for diagnosis of such diseases, has been earnestly desired.
【0004】一方、近年、各種癌組織等において発現し
ているムチンの糖鎖およびコアタンパク質の構造解析が
進展し、癌をはじめとする各種疾患との関連性が注目さ
れてきている〔Bhavanandan, V. P., Glycobiology, 1,
493-503(1991)〕。On the other hand, in recent years, structural analysis of sugar chains and core proteins of mucin expressed in various cancer tissues has progressed, and its relevance to various diseases including cancer has been attracting attention [Bhavanandan, VP, Glycobiology, 1 ,
493-503 (1991)].
【0005】[0005]
【課題を解決するための手段】そこで本発明者らは、上
記課題を解決する目的でヒト胃癌細胞表面に発現する糖
タンパク質に着目して研究をしてきたところ、腫瘍、免
疫異常あるいは各種炎症性疾患の診断への応用が期待さ
れる新規ムチン糖タンパク質39のコアタンパク質をコ
ードする遺伝子を見出し、これが乳癌や膵癌において見
出されたポリモルフィック エピセリアル ムチン(P
EM)と高いホモロジーを示すが、明らかに異なる新し
いムチンであることを明らかにし、本発明を完成した。[Means for Solving the Problems] Therefore, the present inventors have studied for the purpose of solving the above problems by focusing on glycoproteins expressed on the surface of human gastric cancer cells. A gene encoding a core protein of a novel mucin glycoprotein 39, which is expected to be applied to the diagnosis of diseases, was found, and this gene was found in breast cancer and pancreatic cancer, and was found to be polymorphic epithelial mucin (P
The present invention has been completed by revealing that it is a new mucin that clearly shows a high homology with EM).
【0006】すなわち、本発明はヒト由来の新規ムチン
のコアタンパク質をコードする糖タンパク質39遺伝子
を提供するものである。That is, the present invention provides a glycoprotein 39 gene encoding a novel human-derived mucin core protein.
【0007】本発明遺伝子は、例えば配列番号1で示さ
れるアミノ酸配列をコードする塩基配列と配列番号2で
示されるアミノ酸配列をコードする塩基配列、これらの
アミノ酸配列に相補的な塩基配列、又はそれらの両者を
含有するものである。なお、配列表において、塩基配列
の下段は上段の塩基配列より推定されるアミノ酸配列で
ある。The gene of the present invention includes, for example, a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 and a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2, a nucleotide sequence complementary to these amino acid sequences, or a sequence thereof. Both are included. In the sequence listing, the lower part of the base sequence is the amino acid sequence deduced from the upper base sequence.
【0008】本発明の糖タンパク質39遺伝子は、例え
ば以下のようにして調製される。すなわち、まず糖タン
パク質39を発現している細胞より全RNA を分離し、こ
れよりmRNAを精製し、常法によりcDNAを合成したのちこ
れを発現ベクターに組込んだライブラリーを構築する。
次いで抗糖タンパク質39抗体を用いてこのcDNAライブ
ラリーより糖タンパク質39遺伝子を有するクローンを
選択し、本発明の糖タンパク質39遺伝子を得る。次に
上記本発明遺伝子の製法につき、詳細に説明する。The glycoprotein 39 gene of the present invention is prepared, for example, as follows. That is, first, total RNA is isolated from cells expressing glycoprotein 39, mRNA is purified from this, cDNA is synthesized by a conventional method, and then this is incorporated into an expression vector to construct a library.
Next, a clone having the glycoprotein 39 gene is selected from this cDNA library using the anti-glycoprotein 39 antibody to obtain the glycoprotein 39 gene of the present invention. Next, the method for producing the gene of the present invention will be described in detail.
【0009】〔1〕cDNAライブラリーの構築 全RNA の抽出に用いられる組織細胞としてはヒト胃癌組
織又は既にセルラインとして確立された細胞株、例えば
胃癌細胞株KATO-III〔Sekiguchi M., Sakakibara K. an
d Fujii G. (1978). Jpn. J. Exp. Med., 48, p.61-6
8〕が挙げられる。[1] Construction of cDNA Library As tissue cells used for extraction of total RNA, human gastric cancer tissue or cell lines already established as cell lines, for example, gastric cancer cell line KATO-III [Sekiguchi M., Sakakibara K . an
d Fujii G. (1978). Jpn. J. Exp. Med., 48 , p.61-6
8].
【0010】RNA の抽出は、グアニジン−イソチオシア
ネート混合液又は適当な界面活性剤、例えばSDS, NP-4
0、トリトンX-100 、デオキシコール酸等を用いて、或
いはホモジナイザーを用いる方法や凍結融解等の物理的
方法によって、細胞を部分的又は完全に破壊、可溶化し
た後、染色体DNA を、ポリトロン等のミキサーもしくは
注射筒を用い、ある程度せん断し、その後、蛋白質と核
酸分画とを分別する操作により行なわれる。この操作に
は、特にフェノール・クロロホルム抽出もしくは超遠心
を用いるCsCl重層法〔Chirgwin, J. M., et al.,Bioch
emistry, 18, p.5294(1979)〕等が一般に用いられる。RNA is extracted by a guanidine-isothiocyanate mixture or a suitable detergent such as SDS, NP-4.
0, Triton X-100, deoxycholic acid, etc., or by partially or completely disrupting and solubilizing the cells by a homogenizer method or a physical method such as freeze-thawing, the chromosomal DNA is treated with polytron, etc. This is carried out by using a mixer or a syringe described in 1. above, shearing to some extent, and then separating the protein and nucleic acid fractions. For this operation, in particular, the CsCl overlay method using phenol / chloroform extraction or ultracentrifugation [Chirgwin, JM, et al., Bioch
emistry, 18 , p. 5294 (1979)] is generally used.
【0011】また上記各方法においては、RNase による
RNA の分解を防ぐために、RNase インヒビター、例えば
ヘパリン、ポリビニル硫酸、ジエチルピロカーボネー
ト、パナジウム複合体、ベントナイト、マカロイド等を
添加しておくのがよい。In each of the above methods, RNase
In order to prevent RNA degradation, it is advisable to add an RNase inhibitor such as heparin, polyvinylsulfate, diethylpyrocarbonate, a vanadium complex, bentonite and macaloid.
【0012】上記抽出操作に従って得られるRNA からの
mRNAの分離、精製は、抽出物を例えばオリゴdT−セルロ
ース(Colaborative Research Inc.)、ポリU−セファ
ロース(ファルマシア社)等の吸着カラムを用いる方法
により又はバッチ法により実施できる。From RNA obtained according to the above extraction procedure
Separation and purification of mRNA can be carried out by a method using an extract using an adsorption column such as oligo dT-cellulose (Colaborative Research Inc.) or poly U-Sepharose (Pharmacia), or by a batch method.
【0013】上記により得られる精製mRNAは、通常不安
定であり、安定な相補DNA(cDNA) の型に代えられ、目的
遺伝子の増幅を可能とするために微生物由来のベクター
に接続される。インビトロでの、上記mRNAのcDNAへの変
換、即ちcDNAの合成は、一般に次のようにして行なうこ
とができる。The purified mRNA obtained as described above is usually unstable, is replaced by a stable complementary DNA (cDNA) type, and is ligated to a microorganism-derived vector in order to enable amplification of the target gene. The above-mentioned conversion of mRNA into cDNA, that is, synthesis of cDNA in vitro can be generally performed as follows.
【0014】即ち、まずオリゴdTをプライマーとし(こ
のプライマーは遊離のオリゴdTもしくは既にベクタープ
ライマーに付加されたオリゴdTのいずれでもよい)、mR
NAを鋳型としてdNTP(dATP, dGTP, dCTP又はdTTP)の存
在下で、逆転写酵素を用いてmRNAに相補的な一本鎖cDNA
を合成する。次のステップは、上記において遊離のオリ
ゴdTを用いたか、ベクタープライマーに付加されたオリ
ゴdTを用いたかにより、各々以下の如く異なる。That is, first, using oligo dT as a primer (this primer may be either free oligo dT or oligo dT already added to the vector primer), mR
Single-stranded cDNA complementary to mRNA using reverse transcriptase in the presence of dNTP (dATP, dGTP, dCTP or dTTP) using NA as a template
To synthesize. The next steps differ as follows depending on whether the free oligo dT or the oligo dT added to the vector primer was used in the above.
【0015】前者の場合、鋳型としたmRNAをアルカリ処
理等により分解して除去し、その後一本鎖DNA を鋳型と
して逆転写酵素又はDNA ポリメラーゼを用いて二本鎖DN
A を作成する。次に得られる二本鎖DNA の両端をエキソ
ヌクレアーゼで処理し、そのそれぞれに適当なリンカー
DNA 又はアニーリング可能な組合せの塩基を複数付加
し、これを適当なベクターへ組込む。これは使用するベ
クターに応じ公知の方法、例えばヤングらの方法〔Youn
g, R. A. et al., in " DNA Cloning, Vol. 1" ,p.49(1
985) 〕、あるいはグブラーとホフマンの方法〔Gubler,
U. and Hoffman, B. J. Gene, 25, p.263(1983) 〕 な
どを使用して行われる。また、上記cDNAの合成には市販
のcDNA合成キットを用いれば容易に行うことができる。In the former case, the mRNA used as a template is decomposed and removed by alkali treatment or the like, and then the single-stranded DNA is used as a template to form a double-stranded DNA using reverse transcriptase or DNA polymerase.
Create A. Next, treat both ends of the resulting double-stranded DNA with exonuclease,
Multiple bases of DNA or an annealable combination are added, and this is incorporated into an appropriate vector. This is a known method depending on the vector used, such as the method of Young et al. [Youn
g, RA et al., in "DNA Cloning, Vol. 1", p.49 (1
985)], or the method of Gubler and Hoffman [Gubler,
U. and Hoffman, BJ Gene, 25 , p.263 (1983)]. Further, the above-mentioned cDNA can be easily synthesized by using a commercially available cDNA synthesis kit.
【0016】ベクターは、特に制限はされないが、λgt
系のファージベクターやプラスミドベクター等を宿主に
応じて適当に選択し、あるいは組合せて使用できる。こ
こで用いられるベクターとしてはλgt10、λgt11等
を例示でき、λgt10、λgt11をベクターとして用い
る方法は前記ヤングらの方法に準じて行うことができる
。The vector is not particularly limited, but λgt
A system such as a phage vector or a plasmid vector can be appropriately selected depending on the host or used in combination. Examples of the vector used here include λgt10 and λgt11, and the method of using λgt10 and λgt11 as a vector can be performed according to the method of Young et al.
【0017】λgt系のファージベクターに組込んだcDNA
組換え体はインビトロパッケージング液と反応させるこ
とによりcDNA組換え体ファージとなり、λgt10又はλ
gt11のcDNAライブラリーが構築される。上記のλgt系
ファージライブラリーの作成は市販のλgt10又はλgt
11cDNAクローニングキットを用いれば容易に行うこと
ができる。CDNA integrated into a λgt-based phage vector
The recombinant becomes a cDNA recombinant phage by reacting with an in vitro packaging solution, and becomes λgt10 or λgt10.
A gt11 cDNA library is constructed. The above-mentioned λgt-based phage library was prepared using commercially available λgt10 or λgt.
It can be easily performed by using the 11 cDNA cloning kit.
【0018】また、後者の場合、鋳型としてmRNAを残存
させたまま、上記と同様のリンカーを付加した開環状プ
ラスミドと、リンカーDNA(しばしば動物細胞で自立複製
できる領域とmRNAの転写プロモーター領域を含むDNA 断
片が用い得る) とを、アニーリングさせて閉環状とした
後、dTNP存在下で、RNase とDNA ポリメラーゼを共存さ
せてmRNAをDNA 鎖に置換し、完全なプラスミドDNA を作
成できる。In the latter case, the open circular plasmid to which the same linker as above is added while leaving the mRNA as a template and the linker DNA (often containing a region capable of autonomous replication in animal cells and a transcription promoter region of mRNA) Can be used as a DNA fragment) to form a closed circle, and then RNase and DNA polymerase are allowed to coexist in the presence of dTNP to substitute the mRNA for the DNA chain to form a complete plasmid DNA.
【0019】上記の如くして得られるcDNA組換え体プラ
スミドを宿主微生物に導入し、該微生物を形質転換す
る。宿主微生物としては、大腸菌(Escherichia coli)
が代表的であるが、特にこれに限定されず、その他に枯
草菌(Bacillus subtilis)、酵母(Saccharomyces cer
visiae)等も使用することができる。The cDNA recombinant plasmid obtained as described above is introduced into a host microorganism to transform the microorganism. As a host microorganism, Escherichia coli
However, the Bacillus subtilis ( Bacillus subtilis ), yeast ( Saccharomyces cer
visiae ) etc. can also be used.
【0020】DNA の宿主微生物への導入及びこれによる
形質転換の方法としては、一般に用いられている方法、
例えば主として対数増殖期にある細胞を集め、CaCl2 処
理して自然にDNA を取り込みやすい状態にして、プラス
ミドを取り込ませる方法等を採用できる。上記方法にお
いては、通常知られているように形質転換の効率を一層
向上させるためにMgCl2 やRbClを更に共存させることも
できる。また、微生物細胞をスフェロプラスト又はプロ
トプラスト化してから形質転換させる方法も採用するこ
とができる。As a method for introducing DNA into a host microorganism and transforming the same by a generally used method,
For example, a method in which cells in the logarithmic growth phase are mainly collected and treated with CaCl 2 so that DNA can be easily taken up naturally and the plasmid is taken up can be adopted. In the above method, MgCl 2 and RbCl can be further coexisted in order to further improve the efficiency of transformation, as is commonly known. Alternatively, a method in which microbial cells are transformed into spheroplasts or protoplasts and then transformed can also be employed.
【0021】〔2〕糖タンパク質39遺伝子クローンの
選択 上記により得られる形質転換株から、本発明糖タンパク
質39のコアタンパク質をコードするcDNAを含有する株
を選出する方法としては、例えば以下に示す各種方法を
採用できる。[2] Selection of Glycoprotein 39 Gene Clone As a method for selecting a strain containing a cDNA encoding the core protein of the glycoprotein 39 of the present invention from the transformants obtained as described above, for example, the following various types are selected. The method can be adopted.
【0022】(1) 本発明糖タンパク質39を含むレクチ
ン結合糖タンパク質のコアタンパク質に対する抗体を用
いて選出する方法 予め、cDNAを形質転換株内でタンパク質を発現し得るベ
クターに組込み、形質転換株内でタンパク質を産生さ
せ、本発明糖タンパク質39を含むレクチン結合糖タン
パク質のコアタンパク質に対する抗体及び該抗体に対す
る第2抗体を用いて、本発明糖タンパク質39を含むレ
クチン結合糖タンパク質のポリペプチド産生株を検出
し、目的株を得る。(1) Method of selecting using lectin-binding glycoprotein containing glycoprotein 39 of the present invention using an antibody against the core protein: cDNA is previously incorporated into a vector capable of expressing the protein in the transformant, And a second antibody against the core protein of the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention and a second antibody against the antibody to produce a polypeptide producing strain of the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention. Detect and obtain the target strain.
【0023】(2) 動物細胞で本発明糖タンパク質39の
ポリペプチドを産生させてスクリーニングする方法 形質転換株を培養し、遺伝子を増殖させ、その遺伝子を
動物細胞にトランスフェクトし(この場合、自己複製可
能でmRNA転写プロモーター領域を含むプラスミド若しく
は動物細胞染色体にインテグレートするようなプラスミ
ドのいずれでもよい)、遺伝子にコードされたタンパク
質を産生させ、本発明糖タンパク質39を含むレクチン
結合糖タンパク質のコアタンパク質に対する抗体を用い
て本発明糖タンパク質39を含むレクチン結合糖タンパ
ク質のポリペプチドを検出することにより、元の形質転
換株より目的の本発明糖タンパク質39のポリペプチド
部分をコードするcDNAを有する株を選出する。(2) Method of Screening by Producing Polypeptide of Glycoprotein 39 of the Present Invention in Animal Cell A transformed strain is cultured, a gene is grown, and the gene is transfected into an animal cell (in this case, It may be either a plasmid that is replicable and contains an mRNA transcription promoter region or a plasmid that integrates into the chromosome of an animal cell), produces a protein encoded by a gene, and forms a core protein of a lectin-binding glycoprotein containing the glycoprotein 39 of the present invention. By detecting the polypeptide of the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention using an antibody against the above, a strain having a cDNA encoding the polypeptide portion of the target glycoprotein 39 of the present invention from the original transformant strain can be obtained. elect.
【0024】(3) セレクティブ・ハイブリダイゼーショ
ン・トランスレーションの系を用いる方法 形質転換株から得られるcDNAを、ニトロセルロースフィ
ルター等にブロットし、本発明糖タンパク質39を含む
レクチン結合糖タンパク質のポリペプチド産生細胞から
のmRNAをハイブリダイゼーションさせた後、cDNAに対応
するmRNAを回収する。回収されたmRNAを蛋白翻訳系、例
えばアフリカツメガエルの卵母細胞への注入や、ウサギ
網状赤血球ライゼートや小麦胚芽等の無細胞系で蛋白質
に翻訳させ、本発明糖タンパク質39を含むレクチン結
合糖タンパク質のコアタンパク質に対する抗体を用いて
検出して、目的の株を得る。(3) Method Using Selective Hybridization Translation System cDNA obtained from the transformant strain is blotted on a nitrocellulose filter or the like to produce a polypeptide of a lectin-binding glycoprotein containing the glycoprotein 39 of the present invention. After the mRNA from the cells is hybridized, the mRNA corresponding to the cDNA is recovered. A lectin-binding glycoprotein containing the glycoprotein 39 of the present invention containing the glycoprotein 39 of the present invention obtained by injecting the recovered mRNA into a protein translation system, for example, injecting it into oocytes of Xenopus laevis or translating it into a protein in a cell-free system such as rabbit reticulocyte lysate or wheat germ. The target strain is obtained by detection using an antibody against the core protein of.
【0025】なお、上記方法において用いられる本発明
糖タンパク質39を含むレクチン結合糖タンパク質のコ
アタンパク質に対する抗体は、公知の方法により作成す
ることができる。The antibody to the core protein of the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention used in the above method can be prepared by a known method.
【0026】即ち、まず本発明糖タンパク質39を発現
している組織細胞の細胞膜を界面活性剤を用いて可溶化
し、これを糖タンパク質39が結合しうるレクチン結合
アガロースカラムに吸着させて、レクチン結合糖タンパ
ク質を調製する。That is, first, the cell membrane of a tissue cell expressing the glycoprotein 39 of the present invention is solubilized using a surfactant, and this is adsorbed on a lectin-binding agarose column to which the glycoprotein 39 can bind to give a lectin. Prepare the conjugated glycoprotein.
【0027】各種組織細胞の細胞膜可溶化画分分離手段
としては、例えばヒト癌組織、ヒト細胞を適当な緩衝液
中で破砕後、100,000×gの高速遠心分離に付し、その残
渣をトリトン系界面活性剤に溶解し、これを再度100,00
0×gの高速遠心分離に付し、その上清を採取する方法が
挙げられる。As a means for separating cell membrane solubilized fractions of various tissue cells, for example, human cancer tissue and human cells are crushed in an appropriate buffer and subjected to high-speed centrifugation at 100,000 × g, and the residue is triton-based. Dissolve in surfactant and re-add 100,00
A method of subjecting to 0 × g high-speed centrifugation and collecting the supernatant can be mentioned.
【0028】得られた細胞膜可溶化画分より本発明糖タ
ンパク質39を含むレクチン結合糖タンパク質を分離す
るために用いられるレクチンとしては、例えばピーナッ
ツ豆レクチン(Peanut agglutinin, PNA)が挙げられ、
かかるレクチンは市販されているものを用いてもよい
し、例えばピーナッツ豆より自体公知の手段により抽出
分離したものを用いてもよい。レクチン結合アガロース
は市販されているものを用いてもよいし、通常の手段に
よりアガロースゲルにカップリングさせて得ることもで
きる。The lectin used for separating the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention from the obtained cell membrane solubilized fraction includes, for example, peanut bean lectin (Peanut agglutinin, PNA),
As such a lectin, a commercially available one may be used, or, for example, one obtained by extracting and separating from peanut beans by a method known per se may be used. Commercially available lectin-bound agarose may be used, or it may be obtained by coupling it to an agarose gel by a conventional means.
【0029】レクチン結合糖タンパク質の溶出には、ハ
プテン糖、例えばラクトース溶液等が用いられる。ここ
で用いる溶出液の濃度は0.05〜0.2Mが好ましい。To elute the lectin-binding glycoprotein, a hapten sugar such as lactose solution is used. The concentration of the eluate used here is preferably 0.05 to 0.2M.
【0030】次に、本発明糖タンパク質39を含むレク
チン結合糖タンパク質をトリフルオロメタンスルホン酸
(TFMS)またはフッ化水素で処理して糖鎖を除去した
後、これを完全アジュバントと共にウサギ等の小動物に
免疫し、さらに適当な間隔をおいて数回不完全アジュバ
ントと共に免疫した後抗血清を採取する。次に大腸菌を
熱処理し遠心分離して得られる菌体成分と前述で得られ
た抗血清とを4℃にて混和した後、遠心分離すれば求め
るポリクローナル抗体を得ることができる。Next, the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention is treated with trifluoromethanesulfonic acid (TFMS) or hydrogen fluoride to remove the sugar chain, and this is used together with a complete adjuvant in small animals such as rabbits. Antiserum is collected after immunization and several immunizations with incomplete adjuvant at appropriate intervals. Next, the bacterial cell component obtained by heat-treating E. coli and centrifuging and the antiserum obtained above are mixed at 4 ° C. and then centrifuging to obtain the desired polyclonal antibody.
【0031】上記において得られた本発明遺伝子クロー
ンは、常法に従って各種プラスミドにサブクローニング
することができる。例えばEcoRI にて切断して精製した
本発明遺伝子を含むcDNA断片を、同様にEcoRI にて切断
した pUC18〔Yanisch-Perron, C., et al., Gene, 8
3, p.103-119(1985)〕などのクローニングベクターの切
断部位へ挿入すればよい。これにより所望の組換え体プ
ラスミドを得ることができる。また得られる組換え体プ
ラスミドの宿主への導入及びこれによる組換え体プラス
ミドの増幅と個別化は、一般に用いられている各種の方
法、例えば主として対数増殖期にある細胞を集め、CaCl
2 処理により自然にDNA を取り込みやすい状態とし、こ
れをにベクターを取り込ませる方法等により行い得る。The gene clone of the present invention obtained above can be subcloned into various plasmids by a conventional method. For example, a cDNA fragment containing the gene of the present invention purified by digestion with EcoRI was similarly digested with EcoRI to obtain pUC18 [Yanisch-Perron, C., et al., Gene, 8
3 , p.103-119 (1985)] and the like into the cleavage site of the cloning vector. As a result, the desired recombinant plasmid can be obtained. In addition, the introduction of the obtained recombinant plasmid into a host, and the amplification and individualization of the recombinant plasmid thereby, can be performed by various commonly used methods, for example, by collecting cells mainly in logarithmic growth phase and
2 Treatment may be carried out by a method such that the DNA is naturally incorporated into the DNA easily, and the vector is incorporated into the DNA.
【0032】なお、上記において採用される各種の操
作、例えば一部DNA の化学合成、DNA鎖の切断、削除、
付加ないし結合を目的とする酵素処理、DNA の単離、精
製、複製、選択等はいずれも常法に従うことができる。
より具体的には、上記DNA の単離精製は、アガロースゲ
ル電気泳動等により行うことができる。Various operations adopted in the above, for example, chemical synthesis of a part of DNA, cleavage and deletion of DNA chain,
Enzymatic treatment for the purpose of addition or binding, DNA isolation, purification, replication, selection, etc. can all follow conventional methods.
More specifically, the isolation and purification of the above DNA can be performed by agarose gel electrophoresis or the like.
【0033】また、上記で得られる本発明遺伝子の塩基
配列の決定は、適当な制限酵素でDNA を消化した後、ジ
デオキシ法〔Sanger, et al., Proc. Natl. Acad. Sci.
USA, 74, p.5463(1977)〕 やマキサム−ギルバート法
〔A. M. Maxam and W. Gilbert, Methods in Enzymolog
y, 65, p.499(1980)〕等により行い得る。更に上記塩基
配列の決定は、市販のシークエンスキット等を用いるこ
とによっても容易に行い得る。The nucleotide sequence of the gene of the present invention obtained above can be determined by digesting DNA with an appropriate restriction enzyme and then using the dideoxy method [Sanger, et al., Proc. Natl. Acad. Sci.
USA, 74 , p.5463 (1977)] and the Maxam-Gilbert method (AM Maxam and W. Gilbert, Methods in Enzymolog
y, 65 , p. 499 (1980)] and the like. Furthermore, the above-mentioned nucleotide sequence can be easily determined by using a commercially available sequence kit or the like.
【0034】かくして得られた本発明糖タンパク質39
遺伝子の塩基配列の一部及び対応するアミノ酸配列を配
列番号1及び配列番号2に示す。塩基の番号は5′末端
を1とし、5′末端から3′末端方向につけられてい
る。アミノ酸残基の番号はN末端からC末端方向へつけ
られており、最初にコードされるアミノ酸を1としてい
る。配列番号1は配列を決定できた糖タンパク質39遺
伝子の翻訳領域のうち、最も5′末端に位置する180塩
基の長さの翻訳領域で、60個のアミノ酸のタンパク質部
分に相当する。この配列はPEM遺伝子と類似の60塩基(2
0アミノ酸残基)を1単位とするくり返し配列(tandem
repeats)領域と考えられ、くり返し数には個体差があ
ると思われる。また、配列番号2に示される糖タンパク
質39遺伝子の翻訳領域は981塩基の長さで、327個のア
ミノ酸のタンパク質部分に相当する。配列番号1の配列
は塩基配列で3′側(アミノ酸配列でC末端側)にさら
にくり返し配列がつながり、これに配列番号2が接続す
る。The glycoprotein 39 of the present invention thus obtained
A part of the base sequence of the gene and the corresponding amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The base number is 1 at the 5'end and is assigned in the direction from the 5'end to the 3'end. The amino acid residues are numbered from the N-terminal to the C-terminal, and the first encoded amino acid is 1. SEQ ID NO: 1 is a translation region having a length of 180 bases located at the most 5 ′ end in the translation region of the glycoprotein 39 gene whose sequence can be determined, and corresponds to a protein portion of 60 amino acids. This sequence is similar to the PEM gene in 60 bases (2
Repeated sequence (tandem with 0 amino acid residues) as one unit
It is considered to be a repeats) region, and there seems to be individual differences in the number of repeats. The translation region of the glycoprotein 39 gene shown in SEQ ID NO: 2 has a length of 981 bases and corresponds to a protein portion of 327 amino acids. The sequence of SEQ ID NO: 1 is further connected to a repeating sequence on the 3'side (the C-terminal side of the amino acid sequence) in the base sequence, to which the sequence number 2 is connected.
【0035】得られた本発明遺伝子の利用によれば、従
来公知の一般的な遺伝子組換え技術により〔Science, 2
24, p.1431(1984); Biochem. Biophys. Res. Comm., 13
0, p.692(1985); Proc. Natl. Acad. Sci., USA, 80,
p.5990(1983); EP特許公開第187991号公報等参照〕 、
糖タンパク質39のコアタンパク質を容易に且つ大量に
製造、取得することができる。また、このようにして得
られる糖タンパク質39のコアタンパク質を用い、糖タ
ンパク質39のコアタンパク質に特異的な抗体を作成す
ることができる。抗体は通常のポリクローナル抗体、モ
ノクローナル抗体の製造法に従い製造されるが、糖タン
パク質39のコアタンパク質複合体に対するポリクロー
ナル抗体からワインバーガー(Weinberger)らの方法〔Si
ence, 228,p.740-742(1985)〕に従いエピトープ特異的
抗体を得ることも可能である。抗体は糖タンパク質39
及びそのコアタンパク質の精製、測定、識別等に用いら
れる。The obtained gene of the present invention can be used by a conventional publicly known general gene recombination technique [Science, 2
24 , p.1431 (1984); Biochem. Biophys. Res. Comm., 13
0 , p.692 (1985); Proc. Natl. Acad. Sci., USA, 80 ,
p.5990 (1983); see EP Patent Publication No. 187991, etc.),
The core protein of glycoprotein 39 can be easily produced and obtained in large quantities. Further, by using the core protein of glycoprotein 39 thus obtained, an antibody specific to the core protein of glycoprotein 39 can be prepared. Antibodies are produced according to the usual methods for producing polyclonal antibodies and monoclonal antibodies. The polyclonal antibody against the core protein complex of glycoprotein 39 is analyzed by the method of Weinberger et al.
ence, 228 , p.740-742 (1985)], it is also possible to obtain an epitope-specific antibody. Antibody is glycoprotein 39
And its core protein are used for purification, measurement, identification and the like.
【0036】また、上記の如くして得られる糖タンパク
質39のコアタンパク質には、配列表に示すアミノ酸配
列のN末端にメチオニンが結合したポリペプチド、及び
上記アミノ酸配列のN末端に糖タンパク質39のための
シグナルペプチドの部分もしくは全部が結合、又は欠損
した中間体も包含される。かかる変異は天然に、例えば
翻訳後の修飾により得られ、あるいは遺伝子工学的手法
においては、天然から得た遺伝子を例えばサイトスペシ
フィック・ミュータゲネシス等の方法により改変した
り、ホスファイトトリエステル法等の化学合成法により
変異したDNA を合成したり、或いは両者を組合せて、遺
伝子を合成できる。これらの遺伝子を利用し、これを微
生物のベクターに組込み、形質転換された微生物から産
生させることにより、変異を有するコンポーネントを得
ることができる。又、これらのタンパク質は、その機能
を保ったまま、天然或いは人口の変異により、その一部
のアミノ酸の置換や配列の改変を行うことができる。従
って、本発明の糖タンパク質39遺伝子は、上記の各種
変異を有する蛋白質をコードする遺伝子も包含する。遺
伝暗号の末端にはTAG 、TAA 等の終止コドンを付加する
ことができる。遺伝暗号は上記配列番号1及び2に例示
されたコドンに限られず、アミノ酸配列を変えることな
く各アミノ酸に対し任意のコドンを選択でき、例えば遺
伝子組換えに利用する宿主のコドン使用頻度等を考慮し
た常法に従えばよい〔Nucl. Acids. Res., 9, p.43-74
(1981)〕。The glycoprotein 39 core protein obtained as described above includes a polypeptide in which methionine is linked to the N-terminus of the amino acid sequence shown in the sequence listing, and a glycoprotein 39 at the N-terminus of the above amino acid sequence. Also included are intermediates in which part or all of the signal peptide for binding or deficiency is bound. Such a mutation can be obtained naturally, for example, by post-translational modification, or in a genetic engineering technique, a gene obtained from nature is modified by a method such as cytospecific mutagenesis, or a phosphite triester method or the like. Genes can be synthesized by synthesizing mutated DNA by a chemical synthesis method or by combining both. By utilizing these genes, incorporating them into a microbial vector, and producing them from a transformed microorganism, a component having a mutation can be obtained. In addition, these proteins can be partially substituted with amino acids or modified in sequence by natural or artificial mutation while maintaining their functions. Therefore, the glycoprotein 39 gene of the present invention also includes genes encoding proteins having the above-mentioned various mutations. Stop codons such as TAG and TAA can be added to the end of the genetic code. The genetic code is not limited to the codons exemplified in SEQ ID NOS: 1 and 2 above, and any codon can be selected for each amino acid without changing the amino acid sequence. For example, consider the frequency of codon usage of the host used for gene recombination. You can follow the usual method [Nucl. Acids. Res., 9 , p.43-74
(1981)].
【0037】[0037]
【発明の効果】本発明糖タンパク質39遺伝子を用いれ
ば糖タンパク質39のコアタンパク質を容易に且つ大量
に製造することができる。本発明の糖タンパク質39
は、ヒト癌組織、特に胃癌、大腸癌、膵癌、肝癌、食道
癌、肺癌などに発現が認められる一方、胃、結腸、肺な
ど分泌性正常組織に発現していることより、腫瘍マーカ
ー、免疫異常マーカーあるいは各種炎症性疾患マーカー
としての応用が期待される。EFFECT OF THE INVENTION By using the glycoprotein 39 gene of the present invention, the core protein of the glycoprotein 39 can be easily produced in a large amount. Glycoprotein 39 of the present invention
Is expressed in human cancer tissues, particularly gastric cancer, colon cancer, pancreatic cancer, liver cancer, esophageal cancer, lung cancer, etc., while it is expressed in secretory normal tissues such as stomach, colon, lung, etc. It is expected to be applied as an abnormal marker or various inflammatory disease markers.
【0038】[0038]
【実施例】次に実施例を挙げて本発明を更に詳細に説明
する。The present invention will be described in more detail with reference to the following examples.
【0039】実施例1 本発明糖タンパク質39の調製:胃癌細胞KATO-III 2
gを氷冷下CaCl2およびMgCl2添加PBS [PBS(+)] 中で
細断し、これをPotter-Elvehjem 型のホモジナイザーに
てホモジナイズした。Example 1 Preparation of glycoprotein 39 of the present invention: Gastric cancer cell KATO-III 2
g was minced in PBS [PBS (+)] supplemented with CaCl 2 and MgCl 2 under ice cooling and homogenized with a Potter-Elvehjem type homogenizer.
【0040】この破砕液を4℃にて1時間高速遠心(10
5,000×g)し、その上清を除去したペレットに2%トリ
トンX−100 、0.15M NaCl、0.01Mトリス−HCl (pH7.6)
及び50μg/mlのプロテアーゼ阻害剤であるPMSF(フェ
ニルメチルスルホニルフルオライド)(シグマ社)を含
む溶液60mlを加え、さらにこれをホモジナイズしたのち
4℃にて30分放置し、細胞膜を可溶化した。これを4℃
にて1時間高速遠心(105,000×g)し、該上清を得た。This disrupted liquid was centrifuged at 4 ° C. for 1 hour under high speed (10
5,000 × g), and the supernatant was removed, and the pellet was added to 2% Triton X-100, 0.15M NaCl, 0.01M Tris-HCl (pH 7.6).
And 60 ml of a solution containing PMSF (phenylmethylsulfonylfluoride) (Sigma), which is a protease inhibitor of 50 μg / ml, were homogenized, and the mixture was allowed to stand at 4 ° C. for 30 minutes to solubilize the cell membrane. This at 4 ℃
High-speed centrifugation (105,000 × g) for 1 hour to obtain the supernatant.
【0041】該上清を市販のPNA 結合アガロースカラム
(E.Y ラボラトリーズ社製)に添加し、PNA 結合糖タン
パク質を吸着させた。The supernatant was added to a commercially available PNA-binding agarose column (manufactured by EY Laboratories) to adsorb the PNA-binding glycoprotein.
【0042】該カラムを0.1 %トリトンX-100、0.15M N
aCl及び0.01Mトリス-HClを含む洗浄液(pH7.6)200mlにて
洗浄した。その後PNA 結合糖タンパク質を0.05M ラクト
ース溶液50mlにて溶出させた。The column was loaded with 0.1% Triton X-100, 0.15MN.
It was washed with 200 ml of a washing solution (pH 7.6) containing aCl and 0.01 M Tris-HCl. Then, the PNA-bound glycoprotein was eluted with 50 ml of 0.05 M lactose solution.
【0043】溶出液中のPNA 結合糖タンパク質のタンパ
ク濃度はローリー法にて測定し、総量で3mg(タンパク
含量)のPNA 結合糖タンパク質を得た。The protein concentration of PNA-binding glycoprotein in the eluate was measured by the Lowry method to obtain a total amount of 3 mg (protein content) of PNA-binding glycoprotein.
【0044】実施例2 (1) PNA 結合糖タンパク質の糖鎖除去:PNA 結合糖タン
パク質2mgを凍結乾燥後、トリフルオロメタンスルホン
酸(TFMS)−アニソール(2:1)溶液1mlを加えて溶
解した。反応液中に窒素ガスを通気して置換したのち25
℃で5時間撹拌し、糖鎖を分解した。反応終了後、2倍
量のジエチルエーテルを加えて混和したのち、−80℃に
1時間放置した。次に氷冷した50%ピリジン溶液を等量
加えてボルテックスミキサーで撹拌し、次いでエーテル
層を除去した。さらにエーテルを加えて同様にエーテル
抽出を2回行ったのち、2mMピリジン−酢酸バッファー
(pH 5.5)4lに対して、透析した。Example 2 (1) Removal of sugar chain from PNA-bound glycoprotein: 2 mg of PNA-bound glycoprotein was lyophilized and then dissolved by adding 1 ml of trifluoromethanesulfonic acid (TFMS) -anisole (2: 1) solution. Nitrogen gas was bubbled through the reaction solution to replace it.
The mixture was stirred at 0 ° C for 5 hours to decompose sugar chains. After completion of the reaction, a double amount of diethyl ether was added and mixed, and then the mixture was allowed to stand at -80 ° C for 1 hour. Next, an equal volume of ice-cooled 50% pyridine solution was added and stirred with a vortex mixer, and then the ether layer was removed. Ether was further added and the mixture was extracted twice with ether in the same manner, and then dialyzed against 4 l of 2 mM pyridine-acetic acid buffer (pH 5.5).
【0045】(2) PNA 結合糖タンパク質のコアタンパク
質に対するポリクローナル抗体の作成:(1)で調製した
糖鎖除去PNA 結合糖タンパク質のPBS(−)溶液(タン
パク質濃度 800μg/ml)0.5ml とフロインドの完全アジ
ュバント0.5ml を混和して調製した懸濁液をニュージー
ランドホワイト種の雄ウサギの足跂に皮下接種した。そ
の後2週間おきに3回、上記フロインドの不完全アジュ
バントとPNA 結合糖タンパク質の懸濁液を足跂又は背に
皮下接種して免疫した。最終免疫後10日目にウサギの耳
静脈より採血し、完全に凝血させた後4℃で20分間高速
遠心(150,000rpm)を2回くり返して上清を回収し、抗
血清を得た。(2) Preparation of Polyclonal Antibody against Core Protein of PNA-Binding Glycoprotein: 0.5 ml of PBS (-) solution (protein concentration 800 μg / ml) of sugar chain-eliminating PNA-binding glycoprotein prepared in (1) and Freund's The suspension prepared by mixing 0.5 ml of complete adjuvant was subcutaneously inoculated into the foot pad of a New Zealand White male rabbit. Thereafter, the suspension of Freund's incomplete adjuvant and the PNA-binding glycoprotein was subcutaneously inoculated into the foot pad or the back three times every two weeks for immunization. On the 10th day after the final immunization, blood was collected from the ear vein of the rabbit, and after complete coagulation, high-speed centrifugation (150,000 rpm) was repeated twice at 4 ° C. for 20 minutes twice, and the supernatant was collected to obtain antiserum.
【0046】(3) 抗体の吸収処理:後記実施例3(2)で
示す、本発明糖タンパク質39のコアタンパク質をコー
ドする組換え体ファージクローン分離のためのスクリー
ニングに用いる抗体は、大腸菌菌体成分と交差反応しな
いことが望まれる。そこで、予めスクリーニングに用い
る抗体を大腸菌(E. coli Y1090) 菌体成分と反応させ、
これと交差する抗体を除去した。(3) Absorption treatment of antibody: The antibody used in the screening for the isolation of the recombinant phage clone encoding the core protein of the glycoprotein 39 of the present invention shown in Example 3 (2) below is an E. coli cell. It is desired that it not cross-react with the components. Therefore, the antibody used for screening is reacted with E. coli Y1090 bacterial cell components in advance,
Antibodies that crossed this were removed.
【0047】E. coli Y1090 株をLB培地 〔Molecular C
loning (A Laboratory Manual); T.Maniatis, E. F. Fr
itsch, J. Sambrook; Cold Spring Harbor Laboratory
(1982), p.68〕 500ml 中で37℃にて一夜培養し、5000r
pm 、10分間の遠心で菌体を集めた。これを20mlの蒸留
水に懸濁して100 ℃で5〜10分間加熱処理した。更に、
10,000rpm で10分間遠心したのち上清を分離した。次
に、実施例2(2) で作成した抗血清をPBS(−)で50倍
希釈した溶液100mlに、この上清1mlを加えて混和し、
4℃で2時間放置したのち、10,000rpm で15分間遠心
し、その上清を分離して本発明糖タンパク質39のコア
タンパク質に対する抗体を得た。E. coli Y1090 strain was treated with LB medium [Molecular C
loning (A Laboratory Manual); T. Maniatis, EF Fr
itsch, J. Sambrook; Cold Spring Harbor Laboratory
(1982), p.68] Incubate in 500 ml at 37 ℃ overnight,
The cells were collected by centrifugation at pm for 10 minutes. This was suspended in 20 ml of distilled water and heat-treated at 100 ° C for 5-10 minutes. Furthermore,
After centrifugation at 10,000 rpm for 10 minutes, the supernatant was separated. Next, 1 ml of this supernatant was added to 100 ml of a solution prepared by diluting the antiserum prepared in Example 2 (2) 50 times with PBS (-), and mixed.
After leaving it at 4 ° C. for 2 hours, it was centrifuged at 10,000 rpm for 15 minutes, and the supernatant was separated to obtain an antibody against the core protein of glycoprotein 39 of the present invention.
【0048】実施例3 (1) 胃癌細胞株KATO-IIIのcDNAライブラリー作成:胃癌
細胞株KATO-IIIを、RPMI-1640 培地に10%の割合で牛胎
仔血清を加えた培地で5%のCO2 ガス通気下37℃にて継
代培養した。得られた胃癌細胞株KATO-III1gからグア
ニジウムイソチオシアネート法 〔Molecular Cloning
(A Laboratory Manual); T. Maniatis, E. F. Fritsch,
J. Sambrook; Cold Spring Harbor Laboratory (198
2), p.196〕に従って全RNA 3mgを抽出し、これをオリ
ゴ(dT)セルロースカラム(Colaborative Research In
c., カラム容量1ml)を用いてポリ(A)+RNA200μg を得
た。以下アマシャム社のcDNA合成システムのプロトコー
ルに従い、2本鎖のcDNAを合成した。即ち、該当ポリ
(A)+RNA 5μgに逆転写酵素(アマシャム社)を作用さ
せて第一DNA 鎖を合成した。次に大腸菌リボヌクレアー
ゼH(RNase H)及び大腸菌DNA ポリメラーゼI(共にアマ
シャム社)を作用させ、RNA を消化しながら第一DNA 鎖
を鋳型として第二DNA 鎖を合成し、T4DNA ポリメラーゼ
のエキソヌクレアーゼ活性を利用して平滑末端を有する
二本鎖cDNA(ds-cDNA)を合成した。Example 3 (1) Preparation of cDNA library of gastric cancer cell line KATO-III: 5% of gastric cancer cell line KATO-III was added to RPMI-1640 medium at a ratio of 10% fetal calf serum. Subculture was performed at 37 ° C. under aeration of CO 2 gas. From the obtained gastric cancer cell line KATO-III 1 g, the guanidinium isothiocyanate method [Molecular Cloning
(A Laboratory Manual); T. Maniatis, EF Fritsch,
J. Sambrook; Cold Spring Harbor Laboratory (198
2), p.196], 3 mg of total RNA was extracted, and this was extracted with an oligo (dT) cellulose column (Colaborative Research In
c., column volume 1 ml) was used to obtain 200 μg of poly (A) + RNA. Then, double-stranded cDNA was synthesized according to the protocol of the cDNA synthesis system of Amersham. That is, the applicable poly
Reverse transcriptase (Amersham) was allowed to act on 5 μg of (A) + RNA to synthesize the first DNA strand. Next, E. coli ribonuclease H (RNase H) and E. coli DNA polymerase I (both Amersham) are allowed to act, and the second DNA chain is synthesized using the first DNA chain as a template while digesting RNA, and the exonuclease activity of T4 DNA polymerase is A double-stranded cDNA (ds-cDNA) having a blunt end was synthesized by utilizing the method.
【0049】上記により得られたds-cDNA をさらにアマ
シャム社のcDNA・クローニングシステムλgt11を使って
発現ベクターλgt11にクローニングした。即ちds-cDNA
にEcoRI メチラーゼ(アマシャム社)を作用させ、ds-c
DNA の内部にある制限酵素EcoRI の認識部位をメチル基
により保護し、次にT4DNA リガーゼ(アマシャム社)に
より合成EcoRIリンカー(アマシャム社)を両末端に接
続し、最後にこれに制限酵素EcoRI(アマシャム社)を
作用させて両端を付着末端とした。The ds-cDNA obtained above was further cloned into the expression vector λgt11 using the cDNA cloning system λgt11 of Amersham. That is, ds-cDNA
EcoRI methylase (Amersham) is allowed to act on ds-c
The recognition site for the restriction enzyme EcoRI inside the DNA is protected with a methyl group, and then a synthetic EcoRI linker (Amersham) is connected to both ends with T4 DNA ligase (Amersham), and finally the restriction enzyme EcoRI (Amersham) is connected to this. Company) to make both ends sticky ends.
【0050】このds-cDNA とλgt11アーム(アマシャム
社)をT4DNA リガーゼ(アマシャム社)により結合さ
せ、組換えDNA を作成した。これにインビトロパッケー
ジング液(アマシャム社)を作用させてcDNAライブラリ
ーを作成した。This ds-cDNA and λgt11 arm (Amersham) were ligated with T4 DNA ligase (Amersham) to prepare a recombinant DNA. An in vitro packaging solution (Amersham) was allowed to act on this to prepare a cDNA library.
【0051】(2) 本発明糖タンパク質39をコードする
組換え体ファージクローンの分離:(1)で得られたλgt1
1cDNAライブラリーとE. coli Y1090を37℃にて20分間イ
ンキュベートし、組換え体ファージを宿主菌であるY109
0 に吸着させた後、溶解した上層軟寒天と混合して寒天
平板上にまきひろげた。上層寒天固化後寒天平板を42℃
で4〜8時間培養し、プラークを形成させた。次いで10
mMイソプロピル-1-チオ-β-D- ガラクトシド (IPTG) で
飽和させ、乾燥させたニトロセルロースフィルターを寒
天平板表面に置き37℃にて2時間インキュベートして、
β−ガラクトシダーゼ融合タンパク質を発現させた。(2) Isolation of recombinant phage clone encoding the glycoprotein 39 of the present invention: λgt1 obtained in (1)
1 cDNA library and E. coli Y1090 were incubated at 37 ℃ for 20 minutes and recombinant phage was used as host strain Y109.
After being adsorbed at 0, it was mixed with the dissolved upper soft agar and spread on agar plates. After solidifying the upper agar plate, place the agar plate at 42 ° C.
The cells were cultured for 4 to 8 hours to form plaques. Then 10
Saturated with isopropyl-1-thio-β-D-galactoside (IPTG) and dried nitrocellulose filter was placed on the surface of agar plate and incubated at 37 ° C for 2 hours,
The β-galactosidase fusion protein was expressed.
【0052】その後これを4℃にて1時間以上冷却した
後フィルターをはがした。このフィルターを室温で1時
間ブロッキング溶液(2%馬ヘモグロビン、0.1 % Twe
en20、PBS(−))に浸した後、該ブロッキング溶液中
で実施例2(3)で吸収処理した本発明糖タンパク質39
のコアタンパク質に対する抗体50μg/mlと反応させ、室
温にて2時間インキュベートさせた。該フィルターを0.
1% Tween20を含むPBS(−)で5回洗浄後、このフィル
ターをホースラディッシュパーオキシダーゼ(HRP)標
識抗ウサギIgG抗体(Cappel社製)ブロッキング溶液(2
00倍希釈液)中で室温にて2時間反応させ、該反応終了
後、上記の洗浄液で5回洗浄した。次いで過酸化水素含
有4−クロロ−1−ナフトール溶液で発色させて本発明
糖タンパク質39のコアタンパク質に対応する融合タン
パク質を発現しているクローンを選択した。得られたク
ローンの単一プラークを分離した後、Y1090 を宿主とし
て増殖させSM緩衝液中に懸濁させて4℃で保存した。該
クローンをλKP39と命名した。Thereafter, this was cooled at 4 ° C. for 1 hour or more, and then the filter was removed. Apply this filter at room temperature for 1 hour in blocking solution (2% horse hemoglobin, 0.1% Twe).
The glycoprotein 39 of the present invention, which was subjected to the absorption treatment in Example 2 (3) in the blocking solution after being soaked in en20, PBS (-))
Was reacted with 50 μg / ml of an antibody against the core protein of and was incubated at room temperature for 2 hours. Turn the filter to 0.
After washing 5 times with PBS (-) containing 1% Tween20, this filter was washed with horseradish peroxidase (HRP) -labeled anti-rabbit IgG antibody (Cappel) blocking solution (2
The reaction was carried out for 2 hours at room temperature in a (00-fold diluted solution), and after completion of the reaction, the above-mentioned washing solution was washed 5 times. Then, a clone expressing a fusion protein corresponding to the core protein of the glycoprotein 39 of the present invention was selected by coloring with a 4-chloro-1-naphthol solution containing hydrogen peroxide. After separating a single plaque of the obtained clone, it was grown using Y1090 as a host, suspended in SM buffer, and stored at 4 ° C. The clone was named λKP39.
【0053】(3) 本発明糖タンパク質39をコードする
組換え体ファージの溶原菌作成:Huynh, T. V., Young,
R. A., Davis, R. W. :DNA Coloning Vol.1 A Practic
al Approach, (ed.) Glover, D. M., IRL Press(1985)
p.49-78記載の方法に従ってλKP39をE. coli BNN103に
溶原化させた溶原菌を作成した。(3) Preparation of lysogen of recombinant phage encoding glycoprotein 39 of the present invention: Huynh, TV, Young,
RA, Davis, RW: DNA Coloning Vol.1 A Practic
al Approach, (ed.) Glover, DM, IRL Press (1985)
A lysogen was prepared by lysogenizing λKP39 into E. coli BNN103 according to the method described on p.49-78.
【0054】(4) 本発明糖タンパク質39をコードする
組換え体ファージDNAの分離:(2)で得られた本発明糖タ
ンパク質39をコードする組換え体ファージクローン
(λKP39)をE. coli Y1090 を宿主として増殖させたの
ち、〔Molecular Cloning (A Laboratory Manual); T.
Maniatis, E. F. Fritsch, J. Sambrook; ColdSpring H
arbor Laboratory (1982) p.371-372〕記載の方法に従
って、本発明組換え体ファージDNA(λKP39 DNA)を調
製した。(4) Isolation of Recombinant Phage DNA Encoding the Glycoprotein 39 of the Present Invention: The recombinant phage clone (λKP39) encoding the glycoprotein 39 of the present invention obtained in (2) was transformed into E. coli Y1090. After being grown as a host, (Molecular Cloning (A Laboratory Manual); T.
Maniatis, EF Fritsch, J. Sambrook; ColdSpring H
Arbor Laboratory (1982) p.371-372], the recombinant phage DNA of the present invention (λKP39 DNA) was prepared.
【0055】(5) プラスミドpKP39形質転換株の作成:
λKP39 DNAを制限酵素EcoRI(日本ジーン社製)で消化
し、約1900塩基対のDNA断片を得た。(5) Construction of plasmid pKP39 transformant:
λKP39 DNA was digested with restriction enzyme EcoRI (manufactured by Nippon Gene Co., Ltd.) to obtain a DNA fragment of about 1900 base pairs.
【0056】一方、プラスミドベクターpBluescript II
KS(ストラタージーン社製)を同じくEcoRIで消化した
のち、両断片をT4DNAリガーゼ(宝酒造社製)で結合さ
せ、本発明糖タンパク質39のポリペプチド鎖をコード
する組換え体プラスミドpKP39を得た。On the other hand, the plasmid vector pBluescript II
After KS (Stratagene) was similarly digested with EcoRI, both fragments were ligated with T4 DNA ligase (Takara Shuzo) to obtain a recombinant plasmid pKP39 encoding the polypeptide chain of glycoprotein 39 of the present invention. .
【0057】得られた組換え体プラスミドpKP39 をE. c
oli JM83のコンピテント細胞に形質導入した。The obtained recombinant plasmid pKP39 was transformed with E. c.
oli JM83 competent cells were transduced.
【0058】(6) 制限酵素地図の作成:(5)で得られたp
KP39 を 〔Molecular Cloning (A Laboratory Manual);
T. Maniatis, E. F. Fritsch, J. Sambrook; Cold Spr
ing Harbor Laboratory (1982)p.104-106〕 に記載の方
法に従って処理し、さら上記文献p.374-p.381の方法に
従って、本発明糖タンパク質39をコードするpKP39 ク
ローンの制限酵素地図を作成した(図1)。(6) Construction of restriction enzyme map: p obtained in (5)
KP39 (Molecular Cloning (A Laboratory Manual);
T. Maniatis, EF Fritsch, J. Sambrook; Cold Spr
ing Harbor Laboratory (1982) p.104-106], and a restriction enzyme map of the pKP39 clone encoding the glycoprotein 39 of the present invention is prepared according to the method of the above-mentioned documents p.374-p.381. (Fig. 1).
【0059】(7) pKP39 クローンの塩基配列決定:pKP3
9 クローンの塩基配列の決定はサンガー(Sanger)らの
方法 〔Sanger F.,Nicklen S. & Coulson A. R., Proc.
Natl. Acad. Sci. USA, 74, p.5463-5467(1977)〕に従
って行なった。(7) Determination of nucleotide sequence of pKP39 clone: pKP3
9 The nucleotide sequence of the clones was determined by the method of Sanger et al. [Sanger F., Nicklen S. & Coulson AR, Proc.
Natl. Acad. Sci. USA, 74 , p.5463-5467 (1977)].
【0060】以上の結果より得られた糖タンパク質39
遺伝子の配列は、翻訳領域及び3′側の非翻訳領域を含
めて全体で約1900個の塩基からなる。このうち、5′末
端より約600塩基は60塩基を1単位とするくり返し配列
領域である。この領域を含めて翻訳領域は約1560塩基の
長さで、約553個のアミノ酸のタンパク質部分をコード
することが判明した。しかし、くり返し配列領域の配列
は、180塩基(60アミノ酸残基をコードし得る)を決定
できた(配列番号1)が、その他のくり返し配列は未決
定である。くり返し配列より下線の1320塩基の配列は決
定し、配列番号2に示した。Glycoprotein 39 obtained from the above results
The gene sequence is composed of about 1900 bases in total including the translated region and the 3'-untranslated region. Of these, about 600 bases from the 5'end is a repeating sequence region having 60 bases as one unit. It was found that the translation region including this region is about 1560 bases long and encodes a protein portion of about 553 amino acids. However, the sequence of the repetitive sequence region could be determined 180 bases (which can encode 60 amino acid residues) (SEQ ID NO: 1), but other repetitive sequences have not been determined. The sequence of 1320 bases underlined from the repeated sequence was determined and is shown in SEQ ID NO: 2.
【0061】実施例4 (1) 全RNA及びポリ(A)+RNAの調製 実施例3−(1)に示したグアニジウムイソチオシアネー
ト法に従って胃癌細胞株KATO-IIIより全RNA を抽出し、
また市販のオリゴ(dT)セルロースカラム(Colaborati
ve Research Inc.)を用いてポリ(A)+RNA を調製した
(前記MolecularCloning p.196-198 参照)。Example 4 (1) Preparation of total RNA and poly (A) + RNA Total RNA was extracted from gastric cancer cell line KATO-III according to the guanidinium isothiocyanate method described in Example 3- (1),
In addition, a commercially available oligo (dT) cellulose column (Colaborati
Ve Research Inc.) was used to prepare poly (A) + RNA (see Molecular Cloning p. 196-198 above).
【0062】(2) ノーザンブロッティング (1)で調製した全RNA20μg又はポリ(A)+RNA10μgを前記M
olecular Cloning (p.200〜201)の方法に従って、グリ
オキサール存在下、50℃にて1時間加温して変性させた
後、10mMリン酸ナトリウム溶液を含む1%アガロースゲ
ルにて90V で3〜4時間電気泳動を行なった。次に分離
したRNAを20×SSC中でニトロセルロースフィルター(シ
ュライアーアンドシェル社)へ15時間かけて転写させ
た。RNA 転写後のニトロセルロースフィルターを室温で
乾燥後80℃で2時間ベーキングして固定し、その後20mM
トリス塩酸バッファー(pH 8.0)中、100 ℃にて5分間
加熱してグリオキサールを除去した。このフィルターを
実施例3−(7) に記したプレハイブリダイゼーション溶
液中で42℃にて3時間振とうした後、α−32P-dCTP標識
プローブを含むハイブリダイゼーション溶液(組成はプ
ローブ以外プレハイブリダイゼーション溶液と同じ)中
に移して42℃にて20時間振とうした。プローブはpKP39
クローン中cDNAを制限酵素EcoRIで切断した断片をマル
チプライムDNAラベリングシステム(アマシャム社)を
用いてα−32P-dCTPにて標識したものを0.5〜1×107cp
m/mlの濃度で使用した。ハイブリーダイゼーション終了
後、フィルターを2×SSC-0.1%SDS 溶液に移して室温
で10分間ずつ3回洗浄し、更に0.1×SSC-0.1%SDS 溶
液中で60℃にて30分間ずつ3回洗浄した後室温で乾燥し
た。フィルターをろ紙にはりつけてX線フィルムカセッ
トに入れ、X線フィルム(コニカ社XAR-5 )を重ねて−
70℃で1〜3日間感光させた。(2) 20 μg of total RNA prepared in Northern blotting (1) or 10 μg of poly (A) + RNA was added to the above M
After denaturing by heating at 50 ° C for 1 hour in the presence of glyoxal according to the method of olecular Cloning (p.200-201), it was applied to a 1% agarose gel containing 10 mM sodium phosphate solution at 90V for 3-4. Electrophoresis was performed for a period of time. Next, the separated RNA was transferred to a nitrocellulose filter (Schlier and Shell) in 20 × SSC for 15 hours. After RNA transfer, dry the nitrocellulose filter at room temperature, bake at 80 ℃ for 2 hours to fix it, and then fix it at 20 mM.
Glyoxal was removed by heating in Tris-HCl buffer (pH 8.0) at 100 ° C. for 5 minutes. This filter was shaken in the prehybridization solution described in Example 3- (7) at 42 ° C. for 3 hours, and then the hybridization solution containing the α- 32 P-dCTP-labeled probe (composition other than the probe was used. It was transferred to the same as the hybridization solution) and shaken at 42 ° C. for 20 hours. The probe is pKP39
A fragment obtained by cleaving the cDNA of the clone with the restriction enzyme EcoRI was labeled with α- 32 P-dCTP using a multiprime DNA labeling system (Amersham) to give 0.5 to 1 × 10 7 cp.
Used at a concentration of m / ml. After hybridization, transfer the filter to 2 x SSC-0.1% SDS solution and wash 3 times at room temperature for 10 minutes each, and further wash 0.1 x SSC-0.1% SDS solution at 60 ° C for 30 minutes 3 times each. After that, it was dried at room temperature. Put the filter on the filter paper, put it in the X-ray film cassette, and stack the X-ray film (Konica XAR-5)-
It was exposed to light at 70 ° C. for 1 to 3 days.
【0063】得られたノーザンブロッティングの結果を
図2に示す。The obtained northern blotting results are shown in FIG.
【0064】なお、RNA の分子量マーカーとして28S 及
び18S リボゾームRNA を用いた。その結果、4400塩基長
及び6800塩基長の二本のmRNAが検出された。これは既知
のPEM mRNAと同様にオルターナティブ スプライシング
により生じたものと考えられる。28S and 18S ribosomal RNAs were used as RNA molecular weight markers. As a result, two mRNAs having a length of 4400 bases and a length of 6800 bases were detected. It is considered that this is caused by alternative splicing as well as known PEM mRNA.
【0065】[0065]
配列番号:1 配列の長さ:180 配列の型:核酸 鎖の数:二本鎖 トポロジー:直鎖状 配列の種類:cDNA to mRNA 起源 生物名:ホモサピエンス 細胞の種類:胃印環細胞癌 セルライン:KATO-III 直接の起源 ライブラリー名:λgt11 KATO-III cDNA library クローン名:λKP39 配列の特徴 特徴を表す記号:mat peptide 存在位置:1..180 特徴を決定した方法:S 特徴を表す記号:repeat region 存在位置:1..180 特徴を決定した方法:S 特徴を表す記号:repeat unit 存在位置:1..60 特徴を決定した方法:S 配列 GGC TCC ACC GCC CCC CCA GCC CAC GGT GTC ACC TCG GCC CCG GAG AGC 48 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Glu Ser 1 5 10 15 AGG CCG GCC CCG GGC TCC ACC GCG CCC GCA GCC CAC GGT GTC ACC TCG 96 Arg Pro Ala Pro Gly Ser Thr Ala Pro Ala Ala His Gly Val Thr Ser 20 25 30 GCC CCG GAG AGC AGG CCG GCC CCG GGC TCC ACC GCG CCC GCA GCC CAC 144 Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr Ala Pro Ala Ala His 35 40 45 GGT GTC ACC TCG GCC CCG GAC ACC AGG CCG GCC CCG 180 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 50 55 60 SEQ ID NO: 1 Sequence length: 180 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Biological name: Homo sapiens Cell type: Gastric signet ring cell carcinoma Cell Line: KATO-III Direct origin Library name: λgt11 KATO-III cDNA library Clone name: λKP39 Sequence characteristics Characteristic symbol: mat peptide Location: 1..180 Method of determining characteristic: S Characteristic symbol : Repeat region Location: 1..180 Characteristic determination method: S Characteristic symbol: repeat unit Location: 1..60 Characteristic determination method: S Sequence GGC TCC ACC GCC CCC CCA GCC CAC GGT GTC ACC TCG GCC CCG GAG AGC 48 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Glu Ser 1 5 10 15 AGG CCG GCC CCG GGC TCC ACC GCG CCC GCA GCC CAC GGT GTC ACC TCG 96 Arg Pro Ala Pro Gly Ser Thr Ala Pro Ala Ala His Gly Val Thr Ser 20 25 30 GCC CCG GAG AGC AGG CCG GCC CCG GGC TCC ACC GCG CCC GCA GCC CAC 144 Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr Ala Pro Ala Ala His 35 40 45 GGT GTC ACC TCG GCC CCG GAC ACC AGG CCG GCC CCG 180 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 50 55 60
【0066】配列番号:2 配列の長さ:1320 配列の型:核酸 鎖の数:二本鎖 トポロジー:直鎖状 配列の種類:cDNA to mRNA 起源 生物名:ホモサピエンス 細胞の種類:胃印環細胞癌 セルライン:KATO-III 直接の起源 ライブラリー名:λgt11 KATO-III cDNA library クローン名:λKP39 配列の特徴 特徴を表す記号:mat peptide 存在位置:1..981 特徴を決定した方法:S 特徴を表す記号:polyA signal 存在位置:1267..1272 特徴を決定した方法:S 特徴を表す記号:polyA site 存在位置:1293..1320 特徴を決定した方法:S 配列 GGC TCC ACC GCC CCC CCA GCC CAC GGT GTC ACC TCG GCC CCG GAC ACC 48 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 5 10 15 AGG CCC GCC TTG GGC TCC ACC GCG CCT CCA GTC CAC AAT GTC ACC TCG 96 Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser 20 25 30 GCC TCA GGC TCT GCA TCA GGC TCA GCT TCT ACT CTG GTG CAC AAC GGC 144 Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly 35 40 45 ACC TCT GCC AGG GCT ACC ACA ACC CCA GCC AGC AAG AGC ACT CCA TTC 192 Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe 50 55 60 TCA ATT CCC AGC CAC CAC TCT GAT ACT CCT ACC ACC CTT GCC AGC CAT 240 Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His 65 70 75 80 AGC ACC AAG ACT GAT GCC AGT AGC ACT CAC CAT AGC ACG GTA CCT CCT 288 Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Thr Val Pro Pro 85 90 95 CTC ACC TCC TCC AAT CAC AGC ACT TCT CCC CAG TTG TCT ACT GGG GTC 336 Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val 100 105 110 TCT TTC TTT TTC CTG TCT TTT CAC ATT TCA AAC CTC CAG TTT AAT TCC 384 Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser 115 120 125 TCT CTG GAA GAT CCC AGC ACC GAC TAC TAC CAA GAG CTG CAG AGA GAC 432 Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp 130 135 140 ATT TCT GAA ATG TTT TTG CAG ATT TAT AAA CAA GGG GGT TTT CTG GGC 480 Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly 145 150 155 160 CTC TCC AAT ATT AAG TTC AGG CCA GGA TCT GTG GTG GTA CAA TTG ACT 528 Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr 165 170 175 CTG GCC TTC CGA GAA GGT ACC ATC AAT GTC CAC GAC GTG GAG ACA CAG 576 Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln 180 185 190 TTC AAT CAG TAT AAA ACG GAA GCA GCC TCT CGA TAT AAC CTG ACG ATC 624 Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile 195 200 205 TCA GAC GTC AGC GTG AGT GAT GTG CCA TTT CCT TTC TCT GCC CAG TCT 672 Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser 210 215 220 GGG GCT GGG GTG CCA GGC TGG GGC ATC GCG CTG CTG GTG CTG GTC TGT 720 Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys 225 230 235 240 GTT CTG GTT GCG CTG GCC ATT GTC TAT CTC ATT GCC TTG GCT GTC TGT 768 Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys 245 250 255 CAG TGC CGC CGA AAG AAC TAC GGG CAG CTG GAC ATC TTT CCA GCC CGG 816 Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg 260 265 270 GAT ACC TAC CAT CCT ATG AGC GAG TAC CCC ACC TAC CAC ACC CAT GGG 864 Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly 275 280 285 CGC TAT GTG CCC CCT AGC AGT ACC GAT CGT AGC CCC TAT GAG AAG GTT 912 Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val 290 295 300 TCT GCA GGT AAT GGT GGC AGC AGC CTC TCT TAC ACA AAC CCA GCA GTG 960 Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val 305 310 315 320 GCA GCC ACT TCT GCC AAC TTG TAGGGGCACG TCGCCCGCTG AGCTGAGTGG 1011 Ala Ala Thr Ser Ala Asn Leu 325 327 CCAGCCAGTG CCATTCCACT CCACTCAGGT TCTTCAGGGC CAGAGCCCCT GCACCCTGTT 1071 TGGGCTGGTG AGCTGGGAGT TCAGGTGGGC TGCTCACACC GTCCTTCAGA GGCCCCACCA 1131 ATTTCTCGGA CACTTCTCAG TGTGTGGAAG CTCATGTGGG CCCCTGAGGC TCATGCCTGG 1191 GAAGTGTTGT GGTGGGGGCT CCCAGGAGGA CTGGCCCAGA GAGCCCTGAG ATAGCGGGGA 1251 TCCTGAACTG GACTGAATAA AACGTGGTCT CCCACTGCGC CAAAAAAAAA AAAAAAAAAA 1311 AAAAAAAAA 1320SEQ ID NO: 2 Sequence length: 1320 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Homo sapiens Cell type: Gastric signet ring Cellular cancer Cell line: KATO-III Direct origin Library name: λgt11 KATO-III cDNA library Clone name: λKP39 Sequence features Characteristic symbols: mat peptide Location: 1..981 Method of determining features: S features Symbol for expressing: polyA signal Location: 1267..1272 Method for determining feature: S Feature for expressing: polyA site Location: 1293..1320 Method for determining: S sequence GGC TCC ACC GCC CCC CCA GCC CAC GGT GTC ACC TCG GCC CCG GAC ACC 48 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 5 10 15 AGG CCC GCC TTG GGC TCC ACC GCG CCT CCA GTC CAC AAT GTC ACC TCG 96 Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser 20 25 30 GCC TCA GGC TCT GCA TCA GGC TCA GCT TCT ACT CTG GTG CA C AAC GGC 144 Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly 35 40 45 ACC TCT GCC AGG GCT ACC ACA ACC CCA GCC AGC AAG AGC ACT CCA TTC 192 Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe 50 55 60 TCA ATT CCC AGC CAC CAC TCT GAT ACT CCT ACC ACC CTT GCC AGC CAT 240 Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His 65 70 75 80 AGC ACC AAG ACT GAT GCC AGT AGC ACT CAC CAT AGC ACG GTA CCT CCT 288 Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Thr Val Pro Pro 85 90 95 CTC ACC TCC TCC AAT CAC AGC ACT TCT CCC CAG TTG TCT ACT GGG GTC 336 Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val 100 105 110 TCT TTC TTT TTC CTG TCT TTT CAC ATT TCA AAC CTC CAG TTT AAT TCC 384 Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser 115 120 125 TCT CTG GAA GAT CCC AGC ACC GAC TAC TAC CAA GAG CTG CAG AGA GAC 432 Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp 130 135 140 ATT TCT GAA ATG TTT TTG CAG ATT TAT AAA CAA GGG GGT TTT CTG GGC 480 Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly 145 150 155 160 CTC TCC AAT ATT AAG TTC AGG CCA GGA TCT GTG GTG GTA CAA TTG ACT 528 Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr 165 170 175 CTG GCC TTC CGA GAA GGT ACC ATC AAT GTC CAC GAC GTG GAG ACA CAG 576 Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln 180 185 190 TTC AAT CAG TAT AAA ACG GAA GCA GCC TCT CGA TAT AAC CTG ACG ATC 624 Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile 195 200 205 TCA GAC GTC AGC GTG AGT GAT GTG CCA TTT CCT TTC TCT GCC CAG TCT 672 Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser 210 215 220 GGG GCT GGG GTG CCA GGC TGG GGC ATC GCG CTG CTG GTG CTG GTC TGT 720 Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys 225 230 235 240 GTT CTG GTT GCG CTG GCC ATT GTC TAT CTC ATT GCC TTG GCT GTC TGT 768 Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys 245 250 255 CAG TGC CGC CGA AAG AAC TAC GGG CAG CT G GAC ATC TTT CCA GCC CGG 816 Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg 260 265 270 GAT ACC TAC TAC CAT CCT ATG AGC GAG TAC CCC ACC TAC CAC ACC CAT GGG 864 Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly 275 280 285 CGC TAT GTG CCC CCT AGC AGT ACC GAT CGT AGC CCC TAT GAG AAG GTT 912 Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val 290 295 300 TCT GCA GGT AAT GGT GGC AGC AGC CTC TCT TAC ACA AAC CCA GCA GTG 960 Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val 305 310 315 320 GCA GCC ACT TCT GCC AAC TTG TAGGGGCACG TCGCCCGCTG AGCTGAGTGG 1011 Ala Ala Ser Ala Asn Leu 325 327 CCAGCCAGTG CCATTCCACT CCACTCAGGT TCTTCAGGGC CAGAGCCCCT GCACCCTGTT 1071 TGGGCTGGTG AGCTGGGAGT TCAGGTGGGC TGCTCACACC GTCCTTCAGA GGCCCCACCA 1131 ATTTCTCGGA CACTTCTCAG TGTGTGGAAG CTCATGTGGG CCCCTGAGGC TCATGCCTGG 1191 GAAGTGTTGT GGTGGGGGCT CCCAGGAGGA CTGGCCCAGA GAGCCCTGAG ATAGCGGGGA 1251 TCCTGAACTG GACTGAATAA AACGTGGTCT CCCACTGCGC CAAAAAAAAA AAAAAAAAAA 1311 A AAAAAAAA 1320
【図1】本発明糖タンパク質39のコアタンパク質をコ
ードするcDNAの制限酵素地図及び塩基配列決定方法を示
す。図1中、最上段に示したスケールは、cDNAの1番目
の塩基を基準にしたヌクレオチドの長さ(キロベース)
である。その下段は本発明糖タンパク質39をコードす
るcDNAクローンpKP39 を示している。該線上左側の破線
部分は20アミノ酸残基を1単位とするくり返し配列をコ
ードする領域を、中央の太い黒線部分はそれにひき続く
コーディング領域を示す。矢印は各DNA断片について決
定した塩基配列の方向と長さを示す。FIG. 1 shows a restriction enzyme map of a cDNA encoding a core protein of glycoprotein 39 of the present invention and a method for determining a nucleotide sequence. In Figure 1, the scale shown at the top is the length of the nucleotide based on the first base of the cDNA (kilobase).
Is. The lower row shows a cDNA clone pKP39 encoding the glycoprotein 39 of the present invention. The broken line on the left side of the line indicates a region encoding a repetitive sequence with 20 amino acid residues as one unit, and the thick black line in the center indicates the subsequent coding region. Arrows indicate the direction and length of the base sequence determined for each DNA fragment.
【図2】実施例4における本発明タンパク質をコードす
るmRNAのノーザンブロッティングを示す図面である。44
00塩基長及び6800塩基長の2本のmRNAが存在する。FIG. 2 is a drawing showing Northern blotting of mRNA encoding the protein of the present invention in Example 4. 44
There are two mRNAs of 00 base length and 6800 base length.
Claims (3)
ードする塩基配列と配列番号2で示されるアミノ酸配列
をコードする塩基配列とを含有する請求項1記載の遺伝
子。2. The gene according to claim 1, which comprises a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 and a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2.
塩基配列と配列番号2で示される3′末端側部分の塩基
配列とを含有する請求項1記載の遺伝子。3. The gene according to claim 1, which contains the base sequence of the 5'end portion shown in SEQ ID NO: 1 and the base sequence of the 3'end portion shown in SEQ ID NO: 2.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4035085A JP3023469B2 (en) | 1992-02-21 | 1992-02-21 | Glycoprotein 39 gene |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4035085A JP3023469B2 (en) | 1992-02-21 | 1992-02-21 | Glycoprotein 39 gene |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH0751065A true JPH0751065A (en) | 1995-02-28 |
JP3023469B2 JP3023469B2 (en) | 2000-03-21 |
Family
ID=12432134
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP4035085A Expired - Lifetime JP3023469B2 (en) | 1992-02-21 | 1992-02-21 | Glycoprotein 39 gene |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP3023469B2 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000049045A1 (en) * | 1999-02-17 | 2000-08-24 | Hanisch Franz Georg | Peptide variants of the tumour marker muc1 and the use thereof |
WO2005093063A1 (en) * | 2004-03-29 | 2005-10-06 | Medical & Biological Laboratories Co., Ltd. | Kit for solid cancer diagnosis and medicine for solid cancer therapy |
WO2006080192A1 (en) | 2005-01-05 | 2006-08-03 | National University Corporation Chiba University | Gene specific to cancer and diagnosis kit using the same |
EP2169060A1 (en) | 2004-06-02 | 2010-03-31 | alphaGEN Co., Ltd. | Novel polypeptide useful for diagnosis and treatment of cancer |
US7811758B2 (en) | 2004-03-29 | 2010-10-12 | Fujifilm Corporation | Method and apparatus of automatically isolating and purifying nucleic acid |
US7824855B2 (en) | 2004-03-26 | 2010-11-02 | Fujifilm Corporation | Method for selectively separating and purifying RNA and method for separating and purifying nucleic acid |
JP5087925B2 (en) * | 2004-12-07 | 2012-12-05 | 東レ株式会社 | Novel cancer antigen peptides and uses thereof |
US9523091B2 (en) | 2011-07-20 | 2016-12-20 | Sony Corporation | Nucleic-acid extraction method and nucleic-acid extraction cartridge |
-
1992
- 1992-02-21 JP JP4035085A patent/JP3023469B2/en not_active Expired - Lifetime
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000049045A1 (en) * | 1999-02-17 | 2000-08-24 | Hanisch Franz Georg | Peptide variants of the tumour marker muc1 and the use thereof |
AU772709B2 (en) * | 1999-02-17 | 2004-05-06 | Universitat Zu Koln | Peptide variants of the tumour marker MUC1 and the use thereof |
US7824855B2 (en) | 2004-03-26 | 2010-11-02 | Fujifilm Corporation | Method for selectively separating and purifying RNA and method for separating and purifying nucleic acid |
WO2005093063A1 (en) * | 2004-03-29 | 2005-10-06 | Medical & Biological Laboratories Co., Ltd. | Kit for solid cancer diagnosis and medicine for solid cancer therapy |
US7811758B2 (en) | 2004-03-29 | 2010-10-12 | Fujifilm Corporation | Method and apparatus of automatically isolating and purifying nucleic acid |
EP2169060A1 (en) | 2004-06-02 | 2010-03-31 | alphaGEN Co., Ltd. | Novel polypeptide useful for diagnosis and treatment of cancer |
US8222393B2 (en) | 2004-06-02 | 2012-07-17 | Tss Biotech Inc. | Polypeptide useful for cancer diagnosis and treatment |
JP5087925B2 (en) * | 2004-12-07 | 2012-12-05 | 東レ株式会社 | Novel cancer antigen peptides and uses thereof |
WO2006080192A1 (en) | 2005-01-05 | 2006-08-03 | National University Corporation Chiba University | Gene specific to cancer and diagnosis kit using the same |
US7998693B2 (en) | 2005-01-05 | 2011-08-16 | National University Corporation Chiba University | Gene specific to cancer and diagnosis kit using the same |
US9523091B2 (en) | 2011-07-20 | 2016-12-20 | Sony Corporation | Nucleic-acid extraction method and nucleic-acid extraction cartridge |
Also Published As
Publication number | Publication date |
---|---|
JP3023469B2 (en) | 2000-03-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6436703B1 (en) | Nucleic acids and polypeptides | |
US6635742B1 (en) | Antibodies specific for semaphorin-like polypeptides | |
US20080227712A1 (en) | Methods and materials relating to stem cell growth factor-like polypeptides and polynucleotides | |
JP2002501376A (en) | New LDL-receptor | |
US6673904B2 (en) | Stem cell growth factor-like polypeptides | |
US20050192215A1 (en) | Methods and materials relating to novel polypeptides and polynucleotides | |
US20030165921A1 (en) | Novel nucleic acids and polypeptides | |
JP3023469B2 (en) | Glycoprotein 39 gene | |
US20030158400A1 (en) | Novel nucleic acids and polypeptides | |
US6586390B1 (en) | Methods and materials relating to novel prothrombinase-like polypeptides and polynucleotides | |
US20020009786A1 (en) | Novel nucleic acids and polypeptides | |
US20030211987A1 (en) | Methods and materials relating to stem cell growth factor-like polypeptides and polynucleotides | |
US6465620B1 (en) | Methods and materials relating to novel von Willebrand/Thrombospondin-like polypeptides and polynucleotides | |
JP3023467B2 (en) | Human basidin I gene | |
JP3034994B2 (en) | Glycoprotein 10 gene | |
US20020128187A1 (en) | Novel nucleic acids and polypeptides | |
US7544485B2 (en) | Baldness related gene and the polypeptide encoded thereby, and uses | |
US20040059098A1 (en) | Egf motif protein, egfl6 materials and methods | |
US6808890B2 (en) | Method of detecting a cancerous cell expressing EGFL6, and EGF mutif protein | |
US20030170818A1 (en) | Methods and materials relating to novel prothrombinase-like polypeptides and polynucleotides | |
WO2002066600A2 (en) | Methods and materials relating to leukocyte immunoglobulin receptor-like (lir-like) polypeptides and polynucleotides | |
US20030022329A1 (en) | Novel nucleic acids and polypeptides | |
US20030180745A1 (en) | Novel nucleic acids and polypeptides | |
US20030036508A1 (en) | EGF motif protein, EGFL6 materials and methods | |
US20030152941A1 (en) | Nucleic acids encoding novel von Willebrand/Thrombospondin factor-like polypeptides and uses of same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313115 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Year of fee payment: 8 Free format text: PAYMENT UNTIL: 20080121 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090121 Year of fee payment: 9 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Year of fee payment: 9 Free format text: PAYMENT UNTIL: 20090121 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100121 Year of fee payment: 10 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Year of fee payment: 11 Free format text: PAYMENT UNTIL: 20110121 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Year of fee payment: 12 Free format text: PAYMENT UNTIL: 20120121 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Year of fee payment: 13 Free format text: PAYMENT UNTIL: 20130121 |
|
EXPY | Cancellation because of completion of term | ||
FPAY | Renewal fee payment (prs date is renewal date of database) |
Year of fee payment: 13 Free format text: PAYMENT UNTIL: 20130121 |