JPH0584085A - Dna fragment encoding non-a non-b type hepatitis specific antigen, its manifestation and method for detecting non-a non-b type hepatitis virus - Google Patents
Dna fragment encoding non-a non-b type hepatitis specific antigen, its manifestation and method for detecting non-a non-b type hepatitis virusInfo
- Publication number
- JPH0584085A JPH0584085A JP3189268A JP18926891A JPH0584085A JP H0584085 A JPH0584085 A JP H0584085A JP 3189268 A JP3189268 A JP 3189268A JP 18926891 A JP18926891 A JP 18926891A JP H0584085 A JPH0584085 A JP H0584085A
- Authority
- JP
- Japan
- Prior art keywords
- ala
- thr
- gly
- val
- leu
- 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.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、非Ａ非Ｂ型肝炎感染あ
るいは発症時に見られる非Ａ非Ｂ型肝炎特異抗原ポリペ
プチドをコードする新規なＤＮＡ断片に関する。FIELD OF THE INVENTION The present invention relates to a novel DNA fragment encoding a non-A non-B hepatitis-specific antigen polypeptide found at the time of infection or onset of non-A non-B hepatitis.
【０００２】本発明はまた、該ＤＮＡ断片を含む発現ベ
クター、該発現ベクターによって形質転換された形質転
換体および該形質転換体を培養して得られる発現ポリペ
プチドに関する。The present invention also relates to an expression vector containing the DNA fragment, a transformant transformed with the expression vector, and an expression polypeptide obtained by culturing the transformant.
【０００３】本発明はさらに、該ＤＮＡ断片の部分塩基
配列に基づいて合成されたＰＣＲプライマー用一本鎖Ｄ
ＮＡ配列に関する。The present invention further comprises a single-stranded D for PCR primer synthesized on the basis of the partial base sequence of the DNA fragment.
Regarding the NA sequence.
【０００４】本発明はまた、検体中の非Ａ非Ｂ型肝炎ウ
イルス遺伝子及び該ウイルス抗原に対する抗体を検出す
る方法に関する。The present invention also relates to a method for detecting a non-A non-B hepatitis virus gene in a sample and an antibody against the viral antigen.
【０００５】[0005]
【従来の技術】非Ａ非Ｂ型肝炎は伝染性の肝炎であり、
その病因体としてウイルスが示唆されているにも拘ら
ず、患者体内でのウイルス抗原量が少なく、結果的に抗
ウイルス抗体量も少ないためその同定は困難を極めてい
る。このため非Ａ非Ｂ型肝炎の診断は、血清学的には、
アラニンアミノトランスフェラーゼとアスパラギン酸ア
ミノトランスフェラーゼのレベルの上昇を確認し、Ａ型
肝炎、Ｂ型肝炎およびＤ型肝炎、さらには肝障害を引き
起こす既知のウイルス、ＣＭＶ、ＥＢＶ等による肝炎か
否かの診断を行った後、これらに該当しない場合に非Ａ
非Ｂ型肝炎と診断する、いわゆる除外診断法によって行
われていた。かかる繁雑な方法で診断を行うにも拘ら
ず、ＡＬＴ値と非Ａ非Ｂ型肝炎の相関には必然性はな
く、臨床診断のみで非Ａ非Ｂ型肝炎を確定するのは困難
であった。確定診断の手段を欠くため、輸血による、特
に、非Ａ非Ｂ型肝炎健康キャリアーからの供血に起因す
る感染を防止できず、輸血に伴う肝炎の90％以上をこの
肝炎が占めており、その総数は年間100万人にのぼると
いわれている。Non-A non-B hepatitis is a contagious hepatitis,
Although a virus is suggested as the etiological agent, its identification is extremely difficult because the amount of viral antigen in the patient's body is low and the amount of antiviral antibody is consequently low. Therefore, the diagnosis of non-A non-B hepatitis is serologically
We confirmed the elevated levels of alanine aminotransferase and aspartate aminotransferase, and diagnosed whether hepatitis A, hepatitis B and hepatitis D, or known hepatitis caused by liver damage, CMV, EBV, etc. If you don't fall under any of these after
It was performed by a so-called exclusion diagnosis method for diagnosing non-hepatitis B. Despite the diagnosis by such a complicated method, there is no necessity for the correlation between the ALT value and the non-A non-B hepatitis, and it was difficult to determine the non-A non-B hepatitis only by the clinical diagnosis. Due to the lack of a definitive diagnosis method, it is not possible to prevent infection due to blood transfusion, particularly, due to blood donation from non-A non-B hepatitis healthy carriers, and this hepatitis accounts for over 90% of hepatitis associated with blood transfusion. The total number is said to reach one million annually.
【０００６】かかる状況を改善し、非Ａ非Ｂ型肝炎の診
断の確度を上げるために、NIH のAlter によって標準血
清を用いた Alterのパネルが作製され、このAlter のパ
ネルにパスする診断用材料が、ほぼ同時期に、有馬ら
［実験医学，7(2), 196-201(1989) ］とカイロン社のM.
Houghtonら（特表平2-500880号公報）によって開発され
た。有馬らは、患者血清よりＲＮＡを回収し、λｇｔ11
（蛋白質発現ベクター）を用いて患者血清でスクリーニ
ングしている。カイロン社では、患者血漿をチンパンジ
ーに接種して慢性肝炎を起こさせ、高い抗ウイルス抗体
を有する個体から血漿を得、これよりＲＮＡを回収して
λｇｔ11（蛋白質発現ベクター）を用いて患者血清でス
クリーニングしている。カイロン社では、引き続き遺伝
子のクローニングを行い、Ｃ型肝炎ウイルス（ＨＣＶ＝
カイロン社命名）のほぼ全遺伝子のクローニングと、一
部遺伝子を発現して得られた抗原蛋白質のキット化を行
っている。[0006] In order to improve such a situation and improve the accuracy of diagnosis of non-A non-B hepatitis, an Alter panel using standard serum was prepared by Alter of NIH, and a diagnostic material that passes this Alter panel. However, at about the same time, Arima et al. [Experimental Medicine, 7 (2), 196-201 (1989)] and M. Chiron.
Developed by Houghton et al. (Japanese Patent Publication No. 2-500880). Arima et al. Recovered RNA from patient serum and
(Protein expression vector) is used to screen patient sera. Chiron inoculates chimpanzees with patient plasma to cause chronic hepatitis, obtains plasma from individuals with high antiviral antibodies, recovers RNA from this, and screens with patient serum using λgt11 (protein expression vector). is doing. At Chiron, we will continue to clone the gene to obtain the hepatitis C virus (HCV =
Almost all genes (named by Chiron Co., Ltd.) are cloned and the antigenic protein obtained by expressing a part of the genes is made into a kit.
【０００７】しかしながら、未だ疾患の因子は完全には
解明されておらず、また、その数も明らかではない。However, the factors of the disease have not been completely elucidated, and the number thereof is not clear.
【０００８】[0008]
【発明が解決しようとする課題】前述のごとく、Alter
のパネルにパスする診断用材料が開発され、除外診断法
に代わる診断が可能となったが、両者を使用した患者血
清のスクリーニング結果は決して満足できるものではな
く、前者は日本人患者の血清と60〜80％程度しか反応せ
ず、後者は50〜70％程度しか反応しない。即ち、臨床的
に非Ａ非Ｂ型肝炎と診断されているにも拘らず、あるい
は遺伝子増幅（ＰＣＲ）によってＣ型肝炎ウイルス遺伝
子の存在が確認されているにも拘らず、これらの患者血
清と反応しない例が認められる。ウイルスには、その生
存のため、宿主中で変異をおこす機能が備わっているこ
とから、この原因としてカイロン社の単離した遺伝子が
米国人患者由来であり、更にチンパンジー感染という経
路で得られているためチンパンジーに順化した遺伝子に
変異している可能性や、日本人患者に存在するウイルス
遺伝子も単一ではなくかなりの変異を受けている可能性
が高い。[Problems to be Solved by the Invention] As mentioned above, Alter
Although a diagnostic material that passed the panel was developed and it became possible to make an alternative to the exclusion diagnostic method, the screening results of patient sera using both were never satisfactory, and the former was compared with the serum of Japanese patients. Only 60-80% reacts, the latter only 50-70%. That is, despite being clinically diagnosed as non-A non-B hepatitis, or despite the presence of the hepatitis C virus gene being confirmed by gene amplification (PCR), these patient sera There are some cases where no reaction occurs. Since the virus has a function of mutating in the host for its survival, the gene isolated by Chiron is derived from an American patient and is obtained by a route called chimpanzee infection. Therefore, there is a high possibility that it is mutated to a gene that has been adapted to chimpanzees, and that the viral gene present in Japanese patients is not a single gene but is significantly mutated.
【０００９】このため、多数の日本人非Ａ非Ｂ型肝炎患
者から、変異を受けた種々のウイルスＲＮＡを精製・単
離してｃＤＮＡクローンを得、可能な限り多くの患者を
カバーできる反応抗原を、多量に取得することが求めら
れている。Therefore, from various Japanese non-A non-B hepatitis patients, various mutated viral RNAs were purified and isolated to obtain cDNA clones, and reactive antigens capable of covering as many patients as possible were obtained. , Is required to obtain a large amount.
【００１０】さらに、抗体検出系で確定診断のできない
患者血清あるいは感染直後で抗体価の上昇していない血
清では、微量のウイルス遺伝子を検出する遺伝子増幅法
（ＰＣＲ法）での確認が有効である。またこのＰＣＲ法
を用いることにより効率よく遺伝子をクローニングする
ことも可能である。しかしながら、ＰＣＲ法は既知の遺
伝子配列からプライマーを合成し、それを使い反応を行
わせるため、カイロン社の分離したＨＣＶと日本人由来
の非Ａ非Ｂ型肝炎ウイルス遺伝子との間の変異が大きい
場合には、カイロン社由来のＨＣＶ遺伝子の配列をもと
にしたプライマーを使用した場合、変異の大きいウイル
ス遺伝子を検出できない可能性がある。日本人由来の非
Ａ非Ｂ型肝炎ウイルス感染の有無を効率よく検出するた
めには、多くのｃＤＮＡクローンを得、その遺伝子配列
の中で比較的保存されている領域からプライマーを合成
しＰＣＲを行い、特異的にウイルス遺伝子を検出できる
プライマーを探す必要がある。Furthermore, in patient sera that cannot be definitely diagnosed by the antibody detection system or in sera whose antibody titer does not increase immediately after infection, confirmation by a gene amplification method (PCR method) for detecting a trace amount of viral genes is effective. .. It is also possible to efficiently clone a gene by using this PCR method. However, since the PCR method synthesizes a primer from a known gene sequence and uses it to carry out a reaction, the mutation between HCV isolated by Chiron and the non-A non-B hepatitis virus gene of Japanese origin is large. In this case, when a primer based on the sequence of the HCV gene derived from Chiron is used, it is possible that a viral gene with a large mutation cannot be detected. In order to efficiently detect the presence or absence of infection of non-A non-B hepatitis virus of Japanese origin, many cDNA clones were obtained, primers were synthesized from the relatively conserved region in the gene sequence, and PCR was performed. It is necessary to search for primers that can specifically detect viral genes.
【００１１】本発明の目的は、非Ａ非Ｂ型肝炎感染ある
いは発症時に見られるウイルス非構造タンパク質由来の
非Ａ非Ｂ型肝炎特異抗原ポリペプチドをコードする新規
なＤＮＡ断片を提供することである。An object of the present invention is to provide a novel DNA fragment encoding a non-A non-B hepatitis-specific antigen polypeptide derived from a viral non-structural protein that is found during non-A non-B hepatitis infection or onset. ..
【００１２】本発明の別の目的は、該ＤＮＡ断片を含む
発現ベクター、該発現ベクターによって形質転換された
形質転換体、並びに、該形質転換体を培養して得られる
発現ポリペプチドおよびその製造方法を提供することで
ある。Another object of the present invention is to provide an expression vector containing the DNA fragment, a transformant transformed with the expression vector, an expression polypeptide obtained by culturing the transformant, and a method for producing the same. Is to provide.
【００１３】本発明のさらに別の目的は、非Ａ非Ｂ型肝
炎ウイルス遺伝子検出のための合成プライマーを提供す
ることである。Yet another object of the present invention is to provide synthetic primers for the detection of non-A non-B hepatitis virus genes.
【００１４】本発明の他の目的は、検体中の非Ａ非Ｂ型
肝炎ウイルス遺伝子及び該ウイルス抗原に対する抗体を
検出する方法を提供することである。Another object of the present invention is to provide a method for detecting a non-A non-B hepatitis virus gene and an antibody against the viral antigen in a sample.
【００１５】[0015]
【課題を解決するための手段】本発明は、非Ａ非Ｂ型肝
炎患者血漿から直接取得された非Ａ非Ｂ型肝炎ウイルス
ＲＮＡから遺伝子工学的に誘導して得られた、該ウイル
ス非構造タンパク質由来の非Ａ非Ｂ型肝炎特異抗原ポリ
ペプチドをコードする塩基配列を含むＤＮＡ断片を提供
する。The present invention provides a non-A non-B hepatitis virus obtained by genetically inducing from a non-A non-B hepatitis virus RNA directly obtained from plasma of a non-A non-B hepatitis patient. Provided is a DNA fragment containing a nucleotide sequence encoding a non-A non-B hepatitis-specific antigen polypeptide derived from a protein.
【００１６】本発明ＤＮＡ断片の調製にあたっての重要
な特徴は、多数の慢性期にある日本人非Ａ非Ｂ型肝炎患
者の新鮮血漿プールから変異を受けた種々の病因ウイル
ス遺伝子を直接取得したことにある。即ち該血漿プール
から直接非Ａ非Ｂ型肝炎ウイルスＲＮＡを含む全ＲＮＡ
を取り出し、ランダムプライマー法によりｃＤＮＡを合
成し、このｃＤＮＡをλファージに入れｃＤＮＡライブ
ラリーを作製した。さらにこのｃＤＮＡライブラリーを
非Ａ非Ｂ型肝炎患者血清を用いてイムノスクリーニング
し、目的のＤＮＡ断片を得た。得られたＤＮＡ断片をプ
ロープとして用い、複数の慢性期非Ａ非Ｂ型肝炎患者血
漿より得られたｃＤＮＡライブラリーをハイブリダイゼ
ーションアッセイによりスクリーニングし、公知のもの
と相同性の異なる、非Ａ非Ｂ型肝炎患者に特異的なｃＤ
ＮＡを単離した。An important feature in the preparation of the DNA fragment of the present invention is that various mutated pathogenic viral genes were directly obtained from a fresh plasma pool of many Japanese non-A non-B hepatitis patients in chronic stage. It is in. That is, total RNA including non-A non-B hepatitis virus RNA directly from the plasma pool
Was taken out, cDNA was synthesized by the random primer method, and this cDNA was put into λ phage to prepare a cDNA library. Further, this cDNA library was immunoscreened using non-A non-B hepatitis patient sera to obtain a target DNA fragment. Using the obtained DNA fragment as a probe, a cDNA library obtained from a plurality of chronic phase non-A non-B hepatitis patient plasmas was screened by a hybridization assay, and non-A non-B CD specific for hepatitis C patients
NA was isolated.
【００１７】このように、複数の非Ａ非Ｂ型肝炎患者血
漿から直接変異を受けた種々の非Ａ非Ｂ型肝炎ウイルス
ＲＮＡを単離する手法を採用したことは、多数の非Ａ非
Ｂ型肝炎患者の診断および該肝炎ウイルスにより汚染さ
れた血液の検出に高い確度で判定を下し得る免疫学的反
応抗原を提供する途を開くものである。As described above, the fact that the method for isolating various non-A non-B hepatitis virus RNAs directly mutated from the plasmas of a plurality of non-A non-B hepatitis patients was adopted The present invention opens the way to provide immunologically reactive antigens that can be highly accurately determined for the diagnosis of hepatitis C patients and the detection of blood contaminated with the hepatitis virus.
【００１８】以下に、本発明に係るｃＤＮＡライブラリ
ーの調製、ＤＮＡ断片の単離、配列の決定、ポリペプチ
ドの発現、精製単離、並びに酵素抗体法及びＰＣＲ法に
よる非Ａ非Ｂ型肝炎診断への応用について詳細に説明す
る。The preparation of the cDNA library according to the present invention, the isolation of DNA fragments, the determination of the sequence, the expression of the polypeptide, the purification isolation, and the non-A non-B hepatitis diagnosis by the enzyme antibody method and the PCR method are described below. The application to is described in detail.
【００１９】ｃＤＮＡライブラリーの調製 先ず、複数の非Ａ非Ｂ型肝炎患者の新鮮血漿から細胞破
砕物を分離し、その遠心上清を更に遠心によりペレット
化し、このペレットからセシウムトリフルオロアセテー
トを用いる平衡密度勾配遠心法によって全ＲＮＡを沈澱
として分離し、次にフェノール／クロロホルム抽出、エ
タノール沈澱により全ＲＮＡを精製する。Preparation of cDNA Library First, cell lysate is separated from fresh plasma of a plurality of non-A non-B hepatitis patients, the centrifugation supernatant is further pelletized by centrifugation, and cesium trifluoroacetate is used from this pellet. Total RNA is isolated as a precipitate by equilibrium density gradient centrifugation followed by phenol / chloroform extraction and ethanol precipitation to purify the total RNA.
【００２０】得られたＲＮＡ画分を用いて、 Gubler と
Hoffman の方法に従うランダムプライマー法によりｃＤ
ＮＡを合成し、これをＤＮＡメチラーゼ（例えば、EcoR
Ｉメチラーゼ）で処理してメチル化した後に、ＤＮＡリ
ンカー（例えば、EcoRＩリンカー）又はＤＮＡアダプタ
ー（例えば、EcoRＩアダプター）を連結し、このものを
λファージ（例えば、λgt10，λgt11）等のクローニン
グベクターにクローニングし、ｃＤＮＡライブラリーを
作製する。The RNA fraction thus obtained was used for Gubler
CD by random primer method according to Hoffman's method
NA is synthesized, and DNA is synthesized with DNA methylase (eg EcoR
I methylase) and methylated, and then ligated with a DNA linker (for example, EcoRI linker) or a DNA adapter (for example, EcoRI adapter), and this is ligated to a cloning vector such as λ phage (for example, λgt10, λgt11). Clone to create a cDNA library.
【００２１】ＤＮＡ断片の単離および配列決定 次に、λファージｃＤＮＡライブラリーを大腸菌に感染
させ、寒天プレート上で培養してプラークを形成させ
る。プラークをニトロセルロースフィルターに移し取
り、ブロッキングした後、非Ａ非Ｂ型肝炎血清を用いる
イムノスクリーニング法により陽性クローンを検出す
る。あるいは、スクリーニングの効率化を図るため、得
られた陽性クローンをプラスミド等のクローニングベク
ターにクローニングし、ランダムプライマー法により32
Ｐ標識ＤＮＡプローブを作製した後、該プローブを用い
るハイブリダイゼーションアッセイにより前記ｃＤＮＡ
ライブラリーから陽性プラークを検出する。 Isolation and Sequencing of DNA Fragments Next, E. coli is infected with the lambda phage cDNA library and cultured on agar plates to form plaques. The plaques are transferred to a nitrocellulose filter, blocked, and then positive clones are detected by an immunoscreening method using non-A non-B hepatitis serum. Alternatively, in order to improve the efficiency of screening, the obtained positive clones are cloned into a cloning vector such as a plasmid and the 32
After the P-labeled DNA probe is prepared, the cDNA is prepared by a hybridization assay using the probe.
Detect positive plaques from the library.
【００２２】その結果、１３種のクローンが得られ、こ
れらのクローンを、C11-7, C10-11,C10-13, C10-14, C1
0-15, C10-16, C10-17, C10-18, C10-19, C10-21, C10-
22,C10-23 およびC10-35と命名した。As a result, 13 kinds of clones were obtained, and these clones were designated as C11-7, C10-11, C10-13, C10-14, C1.
0-15, C10-16, C10-17, C10-18, C10-19, C10-21, C10-
It was named 22, C10-23 and C10-35.
【００２３】これらのクローンのうち C11-7，C10-11，
C10-13，C10-14，C10-15，C10-16，C10-17，C10-18およ
びC10-19は、各クローンを大腸菌ＨＢ101 株に移入し、
それぞれ形質転換株 E.coli HB101/C11-7 （微工研菌寄
第 11589号），E.coliHB101/C10-11 （微工研菌寄第 11
581号），E.coli HB101/C10-13 （微工研菌寄第 11582
号），E.coli HB101/C10-14 （微工研菌寄第 11583
号）， E.coli HB101/C10-15（微工研菌寄第 11584
号）， E.coli HB101/C10-16（微工研菌寄第 11585
号），E.coli HB101/C10-17 （微工研菌寄第 11586
号）， E. coli HB101/C10-18 （微工研菌寄第 11587
号），E.coli HB101/C10-19 （微工研菌寄第 11588号）
として平成２年７月６日付で寄託され、次いで、これら
の寄託物はブタペスト条約に基づく国際寄託に平成３年
６月１３日付で移管され、微生物工業技術研究所に以下
の受託番号で寄託されている。Among these clones, C11-7, C10-11,
For C10-13, C10-14, C10-15, C10-16, C10-17, C10-18 and C10-19, each clone was transferred to E. coli HB101 strain,
Transformed strains E.coli HB101 / C11-7 (Microtechnological Research Institute No. 11589), E.coli HB101 / C10-11 (Microtechnical Research Institute No. 11589)
No. 581), E. coli HB101 / C10-13 (Microtechnology Research Institute, Microbiology Consortium 11582)
Issue), E. coli HB101 / C10-14
No.), E.coli HB101 / C10-15
Issue), E.coli HB101 / C10-16
No.), E.coli HB101 / C10-17 (Microbiology Research Institute, No. 11586)
Issue), E. coli HB101 / C10-18
No.), E.coli HB101 / C10-19 (Microtechnology Research Institute, No. 11588)
As of July 6, 1990, and then these deposits were transferred to the international deposit under the Budapest Treaty on June 13, 1991, and deposited at the Institute of Microbial Science and Technology with the following deposit numbers. ing.
【００２４】 Ｅ．ｃｏｌｉ ＨＢ１０１ 受託番号（微工研条寄） クローン Ｃ１１−７ 第３４４２号 クローン Ｃ１０−１１ 第３４３４号 クローン Ｃ１０−１３ 第３４３５号 クローン Ｃ１０−１４ 第３４３６号 クローン Ｃ１０−１５ 第３４３７号 クローン Ｃ１０−１６ 第３４３８号 クローン Ｃ１０−１７ 第３４３９号 クローン Ｃ１０−１８ 第３４４０号 クローン Ｃ１０−１９ 第３４４１号 得られた１３クローンのλファージＤＮＡから常法に従
ってｃＤＮＡを取得し、これを適切な制限酵素（例え
ば、EcoRＩ, BamHＩ）で切断し、各ｃＤＮＡ断片をアガ
ロースゲル電気泳動で精製した後、シークエンス用ベク
ターであるＭ13ファージに組み込み、Sangerらのジデオ
キシ鎖終止法［Proc. Natl. Acad. Sci. USA, 74,5463
(1977)］を用いて各ｃＤＮＡ断片の塩基配列を決定す
る。 E. E. coli HB101 accession number ( Microtech Lab.) clone C11-7 No. 3442 clone C10-11 No. 3434 clone C10-13 No. 3435 clone C10-14 No. 3436 clone C10-15 No. 3437 clone C10-16 No. 3438 Clone C10-17 No. 3439 Clone C10-18 No. 3440 Clone C10-19 No. 3441 cDNA was obtained from the obtained 13 clones of λ phage DNA according to a conventional method, and the cDNA was prepared using an appropriate restriction enzyme (for example, , EcoRI, BamHI) and each cDNA fragment was purified by agarose gel electrophoresis and then incorporated into M13 phage, which is a vector for sequencing, and the dideoxy chain termination method of Sanger et al. [Proc. Natl. Acad. Sci. USA, 74,5463
(1977)] to determine the nucleotide sequence of each cDNA fragment.
【００２５】このようにして決定された上記各クローン
の塩基配列及びそれから推定されるアミノ酸配列を配列
表中配列番号１〜１３に示した。即ち、配列番号１〜１
３は夫々、クローン C11-7，C10-11，C10-13，C10-14，
C10-15，C10-16，C10-17，C10-18，C10-19，C10-21，C1
0-22，C10-23およびC10-35について決定された配列を示
す。The nucleotide sequences of the above-mentioned clones thus determined and the amino acid sequences deduced therefrom are shown in SEQ ID NOs: 1 to 13. That is, SEQ ID NOS: 1 to 1
3 are clones C11-7, C10-11, C10-13, C10-14,
C10-15, C10-16, C10-17, C10-18, C10-19, C10-21, C1
The sequences determined for 0-22, C10-23 and C10-35 are shown.
【００２６】13クローンは全て連続的なオープンリーデ
ィングフレーム（読み取り枠）が開き、終止コドンはも
っていなかった。All 13 clones had a continuous open reading frame (open reading frame) open and no stop codon.
【００２７】Ｃ型肝炎ウイルス（ＨＣＶ）はゲノムＲＮ
Ａの解析により日本脳炎ウイルスなどのフラビウイルス
と類似したウイルスであることが知られている（蛋白質
・核酸・酵素35(12)，2117〜2127(1990)）。この公知の
フラビウイルスの遺伝子及びポリペプチドに対する相同
性の比較から、上記クローンはいずれも非Ａ非Ｂ型肝炎
ウイルス非構造タンパク質をコードしているものと考え
られる。Hepatitis C virus (HCV) is a genomic RN
It is known by analysis of A that the virus is similar to flavivirus such as Japanese encephalitis virus (protein / nucleic acid / enzyme 35 (12), 2117 to 2127 (1990)). From the comparison of homology to the gene and polypeptide of this known flavivirus, it is considered that all of the above clones encode a non-A non-B hepatitis virus nonstructural protein.
【００２８】また、これらのクローンの核酸配列および
オープンリーディングフレームに沿って翻訳したアミノ
酸配列をHoughtonら［欧州特許出願第88,310,922.5号お
よび欧州特許出願公開第 318,216号明細書(1988)］によ
り報告されたＣ型肝炎ウイルス(HCV) と比較すると、互
いに相同性があった。即ち、クローン C11-7，C10-16，
C10-17，C10-18，C10-19，C10-21，C10-22およびC10-23
は、HCV と比較的高い相同性を持ち、核酸レベルで80〜
82％，アミノ酸レベルで91〜94％が保存されていた。ま
た、宮村ら［Nuc. Aci. Res., 17, 10367-10372(1989)
］の報告した配列Ｊ１とは更に相同性が高く、核酸は8
5〜95％，アミノ酸は87〜100 ％が保存されていた。こ
れらのクローンはオーバーラップした部分に関し互いに
ホモロジーが高いために第１群に分類した。一方、クロ
ーンC10-11，C10-13，C10-14，C10-15およびC10-35は、
HCV の核酸配列およびJ1の配列と比較すると、核酸レベ
ルでは69〜70％，アミノ酸レベルでは75〜80％の相同性
となり、第１群と比べて相同性が低かった。また、これ
らのクローン間の相同性は高く、互いに保存されている
ため、これを第２群とした。しかし、本発明のＤＮＡ断
片は、特開昭64-2576号公報および特開平1-124387号公
報に開示される非Ａ非Ｂ型肝炎抗原をコードするＤＮＡ
断片との比較においては、核酸レベルでもアミノ酸レベ
ルでも相同性は認められなかった。The nucleic acid sequences of these clones and the amino acid sequences translated along the open reading frame were reported by Houghton et al. [European Patent Application No. 88,310,922.5 and European Patent Application Publication No. 318,216 (1988)]. When compared to hepatitis C virus (HCV), they were homologous to each other. That is, clones C11-7, C10-16,
C10-17, C10-18, C10-19, C10-21, C10-22 and C10-23
Has a relatively high homology with HCV and is
82% and 91 to 94% at the amino acid level were preserved. Also, Miyamura et al. [Nuc. Aci. Res., 17, 10367-10372 (1989)
], And the nucleic acid is 8
5 to 95% and amino acids 87 to 100% were preserved. These clones were classified into the first group because of their high homology with each other regarding the overlapping part. On the other hand, clones C10-11, C10-13, C10-14, C10-15 and C10-35
When compared with the nucleic acid sequence of HCV and the sequence of J1, the homology was 69 to 70% at the nucleic acid level and 75 to 80% at the amino acid level, which was lower than that of the first group. In addition, since the homology between these clones is high and they are conserved with each other, this was designated as the second group. However, the DNA fragment of the present invention is a DNA encoding a non-A non-B hepatitis antigen disclosed in JP-A-64-2576 and JP-A-1-124387.
No homology was observed at the nucleic acid or amino acid level in comparison with the fragments.
【００２９】従って、本発明の実施態様により、非Ａ非
Ｂ型肝炎特異抗原ポリペプチドが、配列番号１〜１３の
いずれかによって示される読み取り枠に従ってコードさ
れるアミノ酸配列の全部又は一部で表わされることを特
徴とする、該ポリペプチドをコードする塩基配列を含む
ＤＮＡ断片を提供する。Thus, according to an embodiment of the present invention, the non-A non-B hepatitis specific antigen polypeptide is represented in whole or in part by the amino acid sequence encoded according to the open reading frame represented by any of SEQ ID NOs: 1-13. A DNA fragment containing a nucleotide sequence encoding the polypeptide is provided.
【００３０】もちろん、これらの塩基配列において、各
アミノ酸に対応する他のコドンから成る他のいかなる塩
基配列も本発明に包含される。Of course, in these base sequences, any other base sequence consisting of other codons corresponding to each amino acid is included in the present invention.
【００３１】このように本発明ＤＮＡ断片は、核酸配列
に関し従来公知のＤＮＡ断片と異なるものであり新規の
ＤＮＡである。非Ａ非Ｂ型肝炎ウイルスは宿主内で変異
を起こし易いために、それを回避するようにＤＮＡの調
製方法等を設定することが重要であり、これにより、後
述するように、本発明ＤＮＡ断片を組み込んだベクター
を発現して得られた発現産物は、公知の発現抗原に勝る
確度で診断を下すことを可能にした。As described above, the DNA fragment of the present invention is a novel DNA which is different from conventionally known DNA fragments in terms of the nucleic acid sequence. Since the non-A non-B hepatitis virus is likely to cause mutation in the host, it is important to set the DNA preparation method and the like so as to avoid it. The expression product obtained by expressing the vector in which the gene was incorporated made it possible to make a diagnosis with higher accuracy than known expression antigens.
【００３２】非Ａ非Ｂ型肝炎特異抗原ポリペプチドの発
現 本発明はまた、本発明ＤＮＡ断片を、プロモーターの下
流に存在するベクター内のクローニング部位に、導入し
て得られる発現ベクターを提供する。ベクターとして
は、プラスミド，ファージ等の慣用のベクタ−が挙げら
れる。 Development of non-A non-B hepatitis-specific antigen polypeptide
The present invention also provides an expression vector obtained by introducing the DNA fragment of the present invention into a cloning site in a vector existing downstream of a promoter. Examples of the vector include common vectors such as plasmids and phages.
【００３３】発現ベクターは、例えば、第１図に示すよ
うにして構築される。発現プラスミド Trp・TrpE・C11-
7 の構築方法を以下に説明する。The expression vector is constructed, for example, as shown in FIG. Expression plasmid Trp / TrpE / C11-
The construction method of 7 is explained below.
【００３４】先ず、前記C11-7 クローンをpUC119に組み
込んで得られたプラスミド pUC・C11-7 DNA を制限酵素
BamHＩおよび ScaＩで消化し、BamHＩ− ScaＩ断片をア
ガロースゲル電気泳動により分離し、グラスパウダー法
により精製する。一方、発現ベクターである Trp・TrpE
DNAをBamHＩ， ScaＩ消化およびバクテリアアルカリ性
ホスファターゼ（ＢＡＰ）処理した後、フェノール抽
出、水層からのエタノール沈澱を行い処理ベクターＤＮ
Ａを得る。このベクターＤＮＡと前述のC11-7 DNA 断片
とを T4 DNA リガーゼの存在下で連結することによっ
て、非Ａ非Ｂ型肝炎特異抗原をコードするＤＮＡを転写
制御できる位置にプロモーターをもつ発現プラスミド T
rp・TrpE・C11-7 を得た。First, the plasmid pUC.C11-7 DNA obtained by incorporating the C11-7 clone into pUC119 was digested with restriction enzymes.
After digestion with BamHI and ScaI, the BamHI-ScaI fragment is separated by agarose gel electrophoresis and purified by the glass powder method. On the other hand, the expression vectors Trp and TrpE
DNA is digested with BamHI and ScaI and treated with bacterial alkaline phosphatase (BAP), followed by phenol extraction and ethanol precipitation from the aqueous layer to obtain the treated vector DN.
Get A. By ligating this vector DNA with the aforementioned C11-7 DNA fragment in the presence of T4 DNA ligase, an expression plasmid T having a promoter at a position capable of transcriptionally controlling the DNA encoding the non-A non-B hepatitis-specific antigen
rp / TrpE / C11-7 was obtained.
【００３５】他のクローンについても適当な制限酵素を
用いて処理し、発現ベクターに組込むことにより、対応
する発現プラスミドを得ることができる。The corresponding expression plasmid can be obtained by treating other clones with an appropriate restriction enzyme and incorporating them into an expression vector.
【００３６】プロモーターとしては、宿主細胞として原
核生物が使用される場合大腸菌、ファージ等由来のも
の、例えばトリプトファン合成酵素オペロン（trp ）、
ラクトースオペロン（lac ）、ラムダファージＰL , Ｐ
R が挙げられ、また宿主細胞として酵母等の真核生物が
使用される場合例えば３−ホスホグリセレートキナーゼ
又は他の解糖系酵素（Holland ら、Biochemistry, 17：
4900(1978) ）に対するプロモーターが利用できる。ま
た、転写終結因子は必ずしも必要ないが、好ましくは発
現ベクター内に存在させるのがよい。When a prokaryote is used as the host cell, the promoter is derived from Escherichia coli, phage or the like, for example, tryptophan synthase operon (trp),
Lactose operon (lac), Lambda phage P L , P
R, and when a eukaryote such as yeast is used as the host cell, for example, 3-phosphoglycerate kinase or other glycolytic enzymes (Holland et al., Biochemistry, 17 :
4900 (1978)) is available. A transcription terminator is not always necessary, but it is preferable that it be present in an expression vector.
【００３７】ベクターはさらに、形質転換された細胞中
での表現型選択を可能にし得るマーカー配列、例えばア
ンピシリン又はテトラサイクリン耐性遺伝子を含有し得
る。The vector may further contain a marker sequence capable of allowing phenotypic selection in transformed cells, for example ampicillin or tetracycline resistance genes.
【００３８】本発明は更に、宿主を、本発明発現ベクタ
ーで形質転換して得られる形質転換体を提供する。宿主
としては、大腸菌，枯草菌，酵母などのこの分野で慣用
される微生物が挙げられるが、動物細胞の使用も可能で
あることは当業者に自明であろう。The present invention further provides a transformant obtained by transforming a host with the expression vector of the present invention. Examples of the host include microorganisms commonly used in this field, such as Escherichia coli, Bacillus subtilis, and yeast, but it will be apparent to those skilled in the art that animal cells can also be used.
【００３９】形質転換は発現ベクターを宿主細胞に移入
するための慣用的方法によって実施される。宿主として
細菌（例えば大腸菌）を用いる場合には、塩化カルシウ
ムを用いる直接取込み法［Mandel,M. とHiga,A., J. Mo
l. Bio. 53, 159-162 (1970)］が使用できる。Transformation is carried out by conventional methods for transferring expression vectors into host cells. When bacteria (eg E. coli) are used as the host, direct uptake method using calcium chloride [Mandel, M. and Higa, A., J. Mo.
l. Bio. 53 , 159-162 (1970)] can be used.
【００４０】さらに、発現ベクターを含む大腸菌等の宿
主を例えばアンピシリン含有２ＹＴ培地に接種し、培養
し、更にアンピシリン含有リン酸培地中で継代培養する
ことによって発現菌体を増殖し、目的ポリペプチドを産
生させることができる。Further, a host such as Escherichia coli containing the expression vector is inoculated into, for example, an ampicillin-containing 2YT medium, cultivated, and further subcultured in an ampicillin-containing phosphate medium to proliferate the expressed bacterial cells to obtain the desired polypeptide Can be produced.
【００４１】組換え非Ａ非Ｂ型肝炎特異抗原ポリペプチ
ドの製造及び精製方法 本発明は、組換え非Ａ非Ｂ型肝炎特異抗原ポリペプチド
の製造方法も提供する。この方法は、本発明の前記ＤＮ
Ａ断片を適当な宿主細胞内で発現させ得る複製可能な発
現ベクターを構築する工程、該発現ベクターを宿主細胞
内に移入して形質転換体を得る工程、該ＤＮＡ断片を発
現させ得る条件下で該形質転換体を培養して組換えポリ
ペプチドを産生させる工程、および該組換えポリペプチ
ドを回収する工程を含む。 Recombinant non-A non-B hepatitis specific antigen polypepti
The present invention also provides a method for producing a recombinant non-A non-B hepatitis-specific antigen polypeptide. This method is based on the DN of the present invention.
A step of constructing a replicable expression vector capable of expressing the A fragment in a suitable host cell, a step of transfecting the expression vector into a host cell to obtain a transformant, under conditions capable of expressing the DNA fragment The step of culturing the transformant to produce a recombinant polypeptide, and the step of recovering the recombinant polypeptide are included.
【００４２】宿主からの目的物の精製方法としては、宿
主細胞を例えば超音波破砕した後に、遠心分離を行って
非Ａ非Ｂ型肝炎ウイルスｃＤＮＡでコードされるポリペ
プチドとTrpEの融合ポリペプチドを含む不溶性画分を
得、この融合ポリペプチドを尿素含有バッファで可溶化
抽出し、イオン交換カラムクロマトグラフィー（例え
ば、 S-Sepharose）に掛けて精製することができる。As a method for purifying the desired product from the host, for example, ultrasonic disruption of the host cell is followed by centrifugation to give a fusion polypeptide of the polypeptide encoded by the non-A non-B hepatitis virus cDNA and TrpE. An insoluble fraction containing the fusion polypeptide can be obtained, and the fusion polypeptide can be solubilized and extracted with a buffer containing urea and subjected to ion exchange column chromatography (for example, S-Sepharose) for purification.
【００４３】従って、本発明はまた、このように発現し
て得られる、配列番号１〜１３によって示されるアミノ
酸配列の全部又は一部を有する組換え非Ａ非Ｂ型肝炎特
異抗原ポリペプチドも提供する。Accordingly, the present invention also provides a recombinant non-A non-B hepatitis-specific antigen polypeptide having the whole or a part of the amino acid sequences shown by SEQ ID NOs: 1 to 13 obtained by such expression. To do.
【００４４】本明細書中、「組換え非Ａ非Ｂ型肝炎特異
抗原ポリペプチド」とは、ベクター内の非Ａ非Ｂ型肝炎
特異抗原ポリペプチドをコードするＤＮＡを発現させて
得られるポリペプチド自体又は該ポリペプチドとシグナ
ルペプチド等の他のペプチドとの融合ポリペプチドを意
味する。In the present specification, the term "recombinant non-A non-B hepatitis specific antigen polypeptide" means a polypeptide obtained by expressing a DNA encoding the non-A non-B hepatitis specific antigen polypeptide in a vector. By itself or a fusion polypeptide of said polypeptide with another peptide such as a signal peptide.
【００４５】非Ａ非Ｂ型肝炎診断への応用 本発明の発現ポリペプチドを SDS−ポリアクリルアミド
ゲル電気泳動に掛けた後、ウェスタンブロット法により
正常人血清又は非Ａ非Ｂ型肝炎患者血清各２検体と抗原
抗体反応させた結果、第２図に示すように該ポリペプチ
ドは患者血清とのみ強く反応することから、この発現ポ
リペプチドは非Ａ非Ｂ型肝炎特異抗原として作用するこ
とが確認された。 Application to Diagnosis of Non-A Non-B Hepatitis After subjecting the expressed polypeptide of the present invention to SDS-polyacrylamide gel electrophoresis, two normal human sera or two non-A non-B hepatitis patient sera by Western blotting As a result of the antigen-antibody reaction with the sample, as shown in FIG. 2, since the polypeptide strongly reacts only with the patient serum, it was confirmed that this expressed polypeptide acts as a non-A non-B hepatitis specific antigen. It was
【００４６】従って、本発明はさらに、非Ａ非Ｂ型肝炎
ウイルス抗原に対する抗体を検出するための免疫学的検
出方法も提供する。この方法は、抗非Ａ非Ｂ型肝炎ウイ
ルス抗体を含む疑いのある検体を、本発明の１種以上の
組換え非Ａ非Ｂ型肝炎特異抗原ポリペプチドと一緒に抗
原−抗体反応を起こす条件下でインキュベーションする
段階、および抗原−抗体複合体を検出する段階、を含
む。Accordingly, the present invention further provides an immunological detection method for detecting an antibody against a non-A non-B hepatitis virus antigen. This method comprises the steps of subjecting a sample suspected of containing an anti-non-A non-B hepatitis virus antibody to an antigen-antibody reaction with one or more recombinant non-A non-B hepatitis specific antigen polypeptides of the invention. Incubating below, and detecting the antigen-antibody complex.
【００４７】該複合体の検出方法としては、酵素抗体測
定法、ラジオイムノアッセイのような慣用技術が使用さ
れる。As a method for detecting the complex, a conventional technique such as an enzyme-antibody assay method and a radioimmunoassay is used.
【００４８】酵素抗体測定法により、発現ポリペプチド
TrpE・C11-7 と市販カイロン社製キット（オーソHCV Ab
ELISAテスト）を用いて非Ａ非Ｂ型肝炎患者血清との反
応の陽性率を比較した結果、カイロン社製キットの場合
７８％の陽性率を与えたのに対して、本発明発現ポリペ
プチドの場合９１％の陽性率を与え、カイロン社製キッ
トに勝る確度と特異性を有していることが判明した（表
１参照）。Expressed polypeptide by enzyme-linked immunosorbent assay
TrpE ・ C11-7 and commercial Chiron kit (Ortho HCV Ab
As a result of comparing the positive rate of the reaction with the serum of non-A non-B hepatitis patients using the ELISA test), the positive rate of 78% was obtained in the case of the kit manufactured by Chiron Co., Ltd. In this case, a positive rate of 91% was given, and it was proved that it had higher accuracy and specificity than the kit manufactured by Chiron (see Table 1).
【００４９】本発明は、さらに配列番号１〜１３によっ
て示される部分塩基配列に基づいて合成されたセンスお
よびアンチセンスを含む塩基配列を用いて、非Ａ非Ｂ型
肝炎ウイルス遺伝子を増幅する遺伝子増幅法を提供す
る。The present invention further comprises a gene amplification for amplifying a non-A non-B hepatitis virus gene using a base sequence containing sense and antisense synthesized on the basis of the partial base sequences represented by SEQ ID NOS: 1 to 13. Provide the law.
【００５０】これらの塩基配列としては、配列番号２、
４、５又は１３によって示される部分塩基配列に基づい
て合成された下記の塩基配列： ５′−ＧＧＡＴＡＣＡＣＣＧＧＴＧＡＣＴＴＴＧＡ−３′（センス）、 ５′−ＴＧＣＡＴＧＣＡＣＧＴＧＧＣＧＡＴＧＴＡ−３′（アンチセンス）、 ５′−ＧＡＴＧＣＣＣＡＣＴＴＣＣＴＣＴＣＣＣＡ−３′（センス）、および ５′−ＧＴＣＡＧＧＧＴＡＡＣＣＴＣＧＴＴＧＧＴ−３′（アンチセンス） を有するＰＣＲプライマー用一本鎖ＤＮＡ配列が挙げら
れ、これらの配列も本発明の範囲内である。一本鎖ＤＮ
Ａ配列中、最初の２つは配列番号５によって示される部
分塩基配列に基づいて、また後の２つはそれぞれ配列番
号２、４、５及び１３、配列番号２、４及び５によって
示される互いに保存された部分塩基配列に基づいて、セ
ンス又はアンチセンス配列となるように合成されたもの
である。The nucleotide sequences of these are SEQ ID NO: 2,
The following nucleotide sequences synthesized on the basis of the partial nucleotide sequences represented by 4, 5 or 13: 5'-GGATATACCCGTGTACTTTGA-3 '(sense), 5'-TGCATGCACGTGGGCGATGTA-3' (antisense), 5'-GATGCCCACTTCCTCTCCCA- Included are single stranded DNA sequences for PCR primers with 3 '(sense), and 5'-GTCAGGGTAACCTCGTTGGT-3' (antisense), and these sequences are also within the scope of the invention. Single chain DN
In the A sequence, the first two are based on the partial base sequence represented by SEQ ID NO: 5, and the latter two are represented by SEQ ID NOS: 2, 4, 5 and 13, respectively, and SEQ ID NOS: 2, 4 and 5, respectively. It was synthesized so as to be a sense or antisense sequence based on the conserved partial base sequence.
【００５１】これらの一本鎖ＤＮＡ配列の合成は、慣用
のＤＮＡ合成法、例えば亜リン酸法、リン酸トリエステ
ル法、固相法等を用いて実施し得る。ＤＮＡ自動合成装
置を利用するのが便利である。The synthesis of these single-stranded DNA sequences can be carried out using a conventional DNA synthesis method such as the phosphite method, the phosphotriester method, the solid phase method and the like. It is convenient to use an automatic DNA synthesizer.
【００５２】本発明ＤＮＡ配列は、ＰＣＲプライマーと
して用いるとき、第２群のウイルス遺伝子に対してより
高い特異性を示す（表２，表３参照）。The DNA sequences according to the invention show a higher specificity for the second group viral genes when used as PCR primers (see Tables 2 and 3).
【００５３】従って、本発明は、検体中の非Ａ非Ｂ型肝
炎ウイルス遺伝子を検出するための方法も提供する。こ
の方法は、検体からＲＮＡを取り出す段階、得られたＲ
ＮＡに逆転写酵素を作用させてｃＤＮＡを合成する段
階、得られたｃＤＮＡに、上記の一本鎖ＤＮＡ配列プラ
イマーを加えてポリメラーゼ連鎖反応を行う段階、およ
び増幅された非Ａ非Ｂ型肝炎ウイルス遺伝子を検出する
段階、を含む。Accordingly, the present invention also provides a method for detecting a non-A non-B hepatitis virus gene in a sample. This method comprises the steps of removing RNA from a sample,
A step of synthesizing cDNA by acting a reverse transcriptase on NA, a step of adding the above-mentioned single-stranded DNA sequence primer to the obtained cDNA, and carrying out a polymerase chain reaction, and an amplified non-A non-B hepatitis virus Detecting the gene.
【００５４】[0054]
【実施例】以下の実施例により、本発明を更に詳細に説
明するが、本発明は本発明の要旨を変えない限りこれら
の実施例に限定されるものではない。The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples unless the gist of the present invention is changed.
【００５５】実施例１非Ａ非Ｂ型肝炎患者血漿からのｃＤＮＡライブラリーの
調製 複数の慢性期にある日本人非Ａ非Ｂ型肝炎患者の新鮮血
漿プールを原料として以下に述べる手順でＲＮＡ画分を
調製後、λｇｔ10，λｇｔ11ファージを用いてｃＤＮＡ
ライブラリーを作製した。Example 1 Preparation of cDNA library from plasma of non-A non-B hepatitis patient
Preparation Using the fresh plasma pools of Japanese non-A non-B hepatitis patients in multiple chronic stages as raw materials, RNA fractions were prepared by the following procedure, and then cDNA was prepared using λgt10 and λgt11 phages.
A library was created.
【００５６】先ず、血漿 5ｌを等量の50mM Tris-HCl(pH
8.0)，1mM EDTAで希釈後、細胞破砕物等を3,500gで20分
間遠心することにより除去し、この上清を更に 45,000r
pm（約100,000g）， 4℃で 4時間遠心することによりペ
レットを得た。このペレットを常法に従い蛋白変性剤で
ある6Mグアニジウムチオシアネートを用いて溶解後、セ
シウムトリフルオロアセテート液の上に上層し、ベック
マン SW50 ローターで33,000rpm ，20℃で18時間遠心し
てペレットを得た。このペレットを10mM Tris-HCl(pH7.
5)，1mM EDTA溶液に溶かし、フェノール：クロロホルム
（１：１）混合液で 2回の抽出操作の後、上清に5M NaC
l を10分の 1量およびエタノールを 2.5倍容量加えて−
20℃に 2時間放置した。 2時間放置後に15,000g で20分
間遠心し、得られたペレットをジエチルピロカーボネー
ト処理水に溶解しRNA 試料とした。First, 5 l of plasma was treated with an equal volume of 50 mM Tris-HCl (pH
8.0), diluted with 1mM EDTA, and then the cell debris etc. are removed by centrifugation at 3,500g for 20 minutes, and the supernatant is further added to 45,000r.
Pellets were obtained by centrifugation at pm (about 100,000 g) for 4 hours at 4 ° C. This pellet was dissolved in a protein denaturant, 6M guanidinium thiocyanate, according to the standard method, then layered on top of the cesium trifluoroacetate solution and centrifuged at 33,000 rpm, 20 ° C for 18 hours in a Beckman SW50 rotor to obtain a pellet. It was This pellet was added to 10 mM Tris-HCl (pH 7.
5), dissolve in 1mM EDTA solution, extract twice with phenol: chloroform (1: 1) mixture, and add 5M NaC to the supernatant.
Add 1/10 volume and 2.5 volumes of ethanol to
It was left at 20 ° C for 2 hours. After leaving it for 2 hours, it was centrifuged at 15,000 g for 20 minutes, and the obtained pellet was dissolved in diethylpyrocarbonate-treated water to give an RNA sample.
【００５７】得られたRNA 試料を用いて、Gubler & Hof
fmanの方法に従い市販キット（Amersham社あるいはBRL
社）を用いランダムプライマー法によりcDNAを合成し
た。合成されたcDNAをEcoRＩメチラーゼで処理した後に
EcoRＩリンカーあるいはEcoRＩアダプターを連結し、λ
ｇｔ10およびλｇｔ11ファージのEcoRＩ部位にクローニ
ングした。作製したcDNAライブラリーは平均１０6 −１
０7 PFU の組換え体ファージを含んでいた。Using the obtained RNA sample, Gubler & Hof
Commercial kit (Amersham or BRL according to the method of fman
Was used to synthesize cDNA by the random primer method. After treating the synthesized cDNA with EcoRI methylase
Connect an EcoRI linker or EcoRI adapter,
It was cloned into the EcoRI site of the gt10 and λgt11 phages. The prepared cDNA library has an average of 10 6 -1
A 0 7 PFU of recombinant phage contained.
【００５８】実施例２非Ａ非Ｂ型肝炎特異的ｃＤＮＡの単離 実施例１で作製したcDNAライブラリーから非Ａ非Ｂ型肝
炎に特異的なcDNAの単離をイムノスクリーニングおよび
ハイブリダイゼーションアッセイにより試みた。Example 2 Isolation of non-A non-B hepatitis-specific cDNA The isolation of cDNA specific to non-A non-B hepatitis from the cDNA library prepared in Example 1 was carried out by immunoscreening and hybridization assay. I tried.
【００５９】最初に、非Ａ非Ｂ型肝炎患者の血清中には
病因ウイルスに特異的な抗体が存在していると考えられ
たので、非Ａ非Ｂ型肝炎患者血清でHBc 抗体およびHBs
抗体陰性のもの２サンプルをプールしλｇｔ11ライブラ
リーのイムノスクリーニングを行った。イムノスクリー
ニングは常法に従って、β−ガラクトシダーゼと融合し
た組換えペプチドと非Ａ非Ｂ型肝炎（NANBH と略称す
る）患者血清との特異的反応を見ることにより行った。First, since it was considered that antibodies specific to the pathogenic virus were present in the serum of non-A non-B hepatitis patients, HBc antibody and HBs were detected in the serum of non-A non-B hepatitis patients.
Two samples of negative antibodies were pooled and immunoscreened with the λgt11 library. The immunoscreening was carried out according to a conventional method by observing a specific reaction between the recombinant peptide fused with β-galactosidase and the serum of a non-A non-B hepatitis (NANBH) patient.
【００６０】先ず、大腸菌Y1090 株とλｇｔ11 cDNA ラ
イブラリーを混ぜ適当な密度で寒天プレートにまき、43
℃で 3時間インキュベートしてプラークを形成させる。
次に、10mM IPTG を染み込ませたHybond-C ニトロセル
ロースフィルターをプレートにかぶせ、37℃で３時間発
現を誘発する。このニトロセルロースフィルターを 3％
ゼラチン溶液を用いてブロッキングした後、NANBH 患者
血清と 4℃で一晩反応させ、洗浄後、さらにパーオキシ
ダーゼ標識抗ヒトIgG （ヤギ抗体）を反応させた。この
フィルターをジアミノベンジジンとＨ2 Ｏ2 との混液と
反応させることにより陽性シグナルが得られた。このク
ローンC11-7 はHBc 抗体、HBs 抗体との反応は認られな
かった。First, Escherichia coli Y1090 strain and λgt11 cDNA library were mixed and spread on an agar plate at an appropriate density.
Incubate at ℃ for 3 hours to allow plaque formation.
Next, a Hybond-C nitrocellulose filter impregnated with 10 mM IPTG is placed on the plate to induce expression at 37 ° C. for 3 hours. 3% of this nitrocellulose filter
After blocking with a gelatin solution, it was reacted with NANBH patient serum at 4 ° C. overnight, washed, and further reacted with peroxidase-labeled anti-human IgG (goat antibody). A positive signal was obtained by reacting this filter with a mixture of diaminobenzidine and H 2 O 2 . This clone C11-7 showed no reaction with HBc antibody or HBs antibody.
【００６１】次に、スクリーニングの効率化を図るた
め、得られたC11-7クローンをpUC119に再びクローニン
グし、ランダムプライマー法でプローブ化し、ハイブリ
ダイゼーションアッセイによりλｇｔ10cDNAライブラリ
ーのスクリーニングを行った。スクリーニングは常法に
従ってC-600hfl(-) 大腸菌を用いて行い、５×１０4 PF
U の組換え体ファージを 150mmＬ−プレートにまく。37
℃で一晩インキュベートし、プラークが出現したら 4℃
に 1時間放置し、更に Hybond-N フィルターをプレート
にかぶせ30秒間放置する。次に、変性溶液(0.5M NaOH,
1.5M NaCl) で湿らせた濾紙の上にこのフィルターをの
せ 1分間放置後、中和溶液（0.5M Tris-HClpH7.0, 1.5M
NaCl）に５分間浸し、更に、 2×SSC 中で洗浄後乾燥
させる。乾燥したフィルターは304nm UVを 2分間照射す
ることによりUV−クロスリンキングした。このフィルタ
−をNANBH 患者血清とのイムノスクリーニングにより得
られたC11-7 クローンからランダムプライマー法により
作製した32Ｐ標識DNA プローブとのハイブリダイゼーシ
ョンによりスクリーニングした。Next, in order to improve the efficiency of the screening, the obtained C11-7 clone was cloned again into pUC119, probed by the random primer method, and the λgt10 cDNA library was screened by the hybridization assay. Screening was carried out using C-600hfl (-) E. coli according to a conventional method, and 5 × 10 4 PF was used.
Plate 150 mm L-plate with U recombinant phage. 37
Incubate at ℃ overnight, 4 ℃ when plaques appear
Let stand for 1 hour, cover with Hybond-N filter on the plate and let stand for 30 seconds. Next, the denaturing solution (0.5 M NaOH,
Place this filter on the filter paper moistened with 1.5M NaCl) and leave it for 1 minute. Then, neutralize the solution (0.5M Tris-HCl pH7.0, 1.5M
NaCl) for 5 minutes, further wash in 2 × SSC and dry. The dried filter was UV-crosslinked by irradiation with 304 nm UV for 2 minutes. This filter was screened by hybridization with a 32 P-labeled DNA probe prepared by the random primer method from the C11-7 clone obtained by immunoscreening with NANBH patient serum.
【００６２】先ず、 1×SSC ，65℃で一晩インキュベー
トした後， 1×SSC ，65℃で10分間ずつ 2回洗浄し、−
70℃でオートラジオグラフィーを行い陽性プラークを検
出する。陽性プラークをSMバッファに取り上げファージ
ストックとした。このスクリーニングにより得られたク
ローンを更に標識プローブとして用い、一連のスクリー
ニングを行った。その結果、あらたに12クローンが得ら
れ、それぞれC10-11，C10-13，C10-14，C10-15，C10-1
6，C10-17，C10-18，C10-19，C10-21，C10-22，C10-23
およびC10-35と命名した。First, after incubating overnight at 1 × SSC and 65 ° C., the plate was washed twice at 1 × SSC and 65 ° C. for 10 minutes each time.
Autoradiography is performed at 70 ° C to detect positive plaques. Positive plaques were picked up in SM buffer and used as phage stock. The clone obtained by this screening was further used as a labeled probe to perform a series of screenings. As a result, 12 new clones were obtained, which were C10-11, C10-13, C10-14, C10-15, and C10-1, respectively.
6, C10-17, C10-18, C10-19, C10-21, C10-22, C10-23
And C10-35.
【００６３】実施例３非Ａ非Ｂ型肝炎特異的ｃＤＮＡの配列決定 得られた１３クローンのλgt11あるいはλgt10のファー
ジDNA からのcDNAを制限酵素EcoRＩまたはBamHＩで切断
し、アガロースゲル電気泳動により精製した後、シーク
エンス用ベクターである、M13 ファージのmp18およびmp
19(Messing, J., Methods in Enzymology, 101,20-78)
またはpUC118,119(Vieira,J.とMessing,J., Methods in
Enzymology,153,3-11) に組み込み、Sangerらのジデオ
キシ鎖終止法を用いて配列決定した。各クローンの塩基
配列および推定アミノ酸配列を配列表中の配列番号１〜
１３に示した。即ち、配列番号１〜１３は夫々、クロー
ンC11-7, C10-11, C10-13, C10-14, C10-15, C10-16, C
10-17, C10-18, C10-19, C10-21, C10-22, C10-23 およ
びC10-35について決定された配列を示す。Example 3 Sequencing of non-A non-B hepatitis-specific cDNA The cDNA from the 13 clones of λgt11 or λgt10 phage DNA obtained was cleaved with restriction enzymes EcoRI or BamHI and purified by agarose gel electrophoresis. Then, M13 phage mp18 and mp
19 (Messing, J., Methods in Enzymology, 101, 20-78)
Or pUC118,119 (Vieira, J. and Messing, J., Methods in
Enzymology, 153, 3-11) and sequenced using the dideoxy chain termination method of Sanger et al. The nucleotide sequence and deduced amino acid sequence of each clone are shown in SEQ
13 shows. That is, SEQ ID NOS: 1 to 13 are clones C11-7, C10-11, C10-13, C10-14, C10-15, C10-16, C, respectively.
The sequences determined for 10-17, C10-18, C10-19, C10-21, C10-22, C10-23 and C10-35 are shown.
【００６４】実施例４非Ａ非Ｂ型肝炎ウイルスｃＤＮＡでコードされるポリペ
プチドの発現および精製 （ｉ）発現プラスミドの構築 得られたクローンの一つである C11-7を大腸菌(E.coli)
内で Trpプロモーター下にTrpEとの融合ポリペプチドと
して発現させた。Example 4 Polype encoded by non-A non-B hepatitis virus cDNA
Expression and purification of peptide (i) Construction of expression plasmid One of the obtained clones, C11-7, was transformed into E. coli.
It was expressed as a fusion polypeptide with TrpE under the Trp promoter.
【００６５】先ず、C11-7 クローンをpUC119に組み込ん
で得られたプラスミド pUC・C11-7DNA １μｇを制限酵
素反応液20μｌ［150mM NaCl, 6mM Tris-HCl(pH7.9) ，
6mMMgCl2 ，15単位のBamHＩ酵素および15単位の ScaＩ
酵素］中で37℃、１時間消化反応を行い、その後 0.8％
アガロースゲル電気泳動を行って約700bp のBamHＩ−Sc
aＩ断片を分離し、これをグラスパウダー法（Ｇｅｎｅ
ＣｌｅａｎTM，Ｂｉｏ−１０１社）により精製した。First, 1 μg of the plasmid pUC.C11-7DNA obtained by incorporating the C11-7 clone into pUC119 was added with 20 μl of a restriction enzyme reaction solution [150 mM NaCl, 6 mM Tris-HCl (pH 7.9),
6mMMgCl 2 , 15 units of BamHI enzyme and 15 units of ScaI
Enzyme] at 37 ℃ for 1 hour, then 0.8%
About 700 bp BamHI-Sc after agarose gel electrophoresis
The aI fragment was isolated and this was isolated by the glass powder method (Gene
Clean ™ , Bio-101).
【００６６】発現ベクターである Trp・TrpEのDNA １μ
ｇを反応液20μｌ［150mM NaCl, 6mM Tris-HCl(pH7.
5), 6mM MgCl2 , 15単位のBamHＩ酵素および15単位の S
caＩ酵素］中37℃で 1時間消化し、その反応液に水39μ
ｌを加え、70℃で 5分間熱処理した後にバクテリアアル
カリ性ホスファターゼ(BAP) １μｌ（250単位／μｌ）
を加えて37℃で 1時間保温した。この反応液にフェノー
ルを加えてフェノール抽出を行い、得られた水層をエタ
ノール沈澱し、沈澱物を乾燥した。得られたBamHＩ− S
caＩ処理ベクターDNA １μｇと上述のC11-7 DNA 断片を
10×リガーゼ用バッファ［660mM Tris-HCl(pH7.5), 66m
M MgCl2 , 100mM ジチオスレイトール，1mM ATP ］ 5μ
ｌ，T4 DNAリガーゼ 1μｌ（350 単位／μｌ）に水を加
えて50μｌとし、16℃で一晩保温し連結反応を行った。Expression vector Trp / TrpE DNA 1 μ
20 g of the reaction solution [150 mM NaCl, 6 mM Tris-HCl (pH 7.
5), 6 mM MgCl 2 , 15 units of BamHI enzyme and 15 units of S
digested with caI enzyme] at 37 ℃ for 1 hour and added 39μ of water to the reaction mixture.
1 μl, heat treated at 70 ℃ for 5 minutes, and then bacterial alkaline phosphatase (BAP) 1 μl (250 units / μl)
Was added and the mixture was incubated at 37 ° C for 1 hour. Phenol was added to this reaction solution for phenol extraction, the obtained aqueous layer was ethanol-precipitated, and the precipitate was dried. The obtained BamHI-S
1 μg of caI treated vector DNA and the above C11-7 DNA fragment
10 x ligase buffer [660mM Tris-HCl (pH7.5), 66m
M MgCl 2 , 100 mM dithiothreitol, 1 mM ATP] 5 μ
l, T4 DNA ligase 1 μl (350 units / μl) was added with water to make 50 μl, and incubated at 16 ° C. overnight for ligation reaction.
【００６７】この反応液の10μｌを用いて大腸菌HB101
株を形質転換した。形質転換に用いる感受性大腸菌株
は、塩化カルシウム法［Mandel,M. とHiga,A.,J.Mol.Bi
ol. 53,159-162(1970)］により作られる。形質転換した
大腸菌を25μｇ／mlのアンピシリンを含むLB−プレート
(1％トリプトン，0.5％酵母エキス，0.5% NaCl, 1.5％
寒天）上に塗布し、37℃に一晩保温した。プレート上に
生じた菌のコロニーを一白金耳取り、25μｇ／mlアンピ
シリンを含むＬＢ培地に移し、一晩37℃で培養した。
1.5mlの菌培養液を遠心して集菌し、プラスミドDNA の
ミニプレパレーションをアルカリ法（Maniatisら，Mole
cular Cloning ：A Laboratory Manual,1982）により行
った。得られたプラスミドDNA １μｇを反応液20μｌ
［150mM NaCl,6mM Tris-HCl(pH7.5), 6mM MgCl2 , 15
単位のBamHＩおよび ScaＩ酵素］中で37℃， 1時間消化
し、アガロースゲル電気泳動を行って、700bp のBamHＩ
− ScaＩ断片が生じる Trp・TrpE・C11-7 発現プラスミ
ドを選別した。このプラスミドは、大腸菌 HB101株に移
入され形質転換株 E.coli HB101/Trp・TrpE・C11-7
（微工研菌寄第 11590号）として平成２年７月６日付で
寄託され、次いで、この寄託物はブダペスト条約に基づ
く国際寄託に平成３年６月１３日付で移管され、微生物
工業技術研究所に微工研条寄第３４４３号の受託番号で
寄託されている。E. coli HB101 was prepared using 10 μl of this reaction solution.
The strain was transformed. The susceptible E. coli strains used for transformation were the calcium chloride method [Mandel, M. and Higa, A., J. Mol. Bi.
ol. 53, 159-162 (1970)]. LB-plate containing 25 μg / ml ampicillin of transformed E. coli
(1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1.5%
It was spread on agar and kept at 37 ° C overnight. One platinum loop was taken from the colony of the bacteria produced on the plate, transferred to an LB medium containing 25 μg / ml ampicillin, and cultured overnight at 37 ° C.
1.5 ml of the bacterial culture was centrifuged to collect the cells, and the mini-preparation of plasmid DNA was performed by the alkaline method (Maniatis et al., Mole.
cular Cloning: A Laboratory Manual, 1982). 1 μg of the obtained plasmid DNA 20 μl of reaction solution
[150 mM NaCl, 6 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 , 15
Unit of BamHI and ScaI enzyme] at 37 ° C for 1 hour and subjected to agarose gel electrophoresis to obtain 700 bp BamHI
-A Trp / TrpE / C11-7 expression plasmid producing a ScaI fragment was selected. This plasmid was transferred into E. coli HB101 strain and transformed into E. coli HB101 / Trp / TrpE / C11-7.
(Ministry of Microbiology Research No. 11590) was deposited on July 6, 1990, and then this deposit was transferred to an international deposit under the Budapest Treaty on June 13, 1991, to study microbial technology. It has been deposited with the deposit number of Micromachine Research Article No. 3443.
【００６８】（ii）クローンC11-7 でコードされるポリ
ペプチドの発現および精製 発現プラスミド Trp・TrpE・C11-7 をもつ大腸菌HB101
株を50μｇ／mlのアンピシリンを含む３mlの2YT 培地
(1.6％トリプトン， 1％酵母エキス， 0.5％NaCl）に接
種し、37℃で 9時間培養する。この培養液 1mlを50μｇ
／mlのアンピシリンを含む 100mlのM9-CA 培地(0.6% Ｎ
ａ2 ＨＰＯ4 ， 0.5% ＫＨ2 ＰＯ4 ， 0.5％ ＮａＣ
ｌ， 0.1％ ＮＨ4 Ｃｌ，0.1mM ＣａＣｌ2 ，2mM Ｍｇ
ＳＯ4 ， 0.5％カザミノ酸， 0.2％グルコース）に植え
継ぎ、37℃で21時間培養した。更に、本培養液18mlを
1.2ｌのM9-CA 培地に植え継ぎ、37℃で培養した。ＯＤ
600 ＝0.3 のときに終濃度40mg／ｌになるようにインド
ールアクリル酸を加え、更に16時間培養した。この培養
液を遠心分離して菌体を集めた。菌体に20mlのバッファ
A（50mM Tris-HCl (pH8.0), 1mM EDTA ,30mM NaCl）を
加えて懸濁し、再び遠心分離を行って発現菌体 2.6ｇを
得た。得られた菌体をバッファA 10ml中に懸濁し、超音
波破砕により大腸菌膜を破砕した後に遠心分離を行い、
非Ａ非Ｂ型肝炎ウイルスｃＤＮＡでコードされるポリペ
プチドとTrpEとの融合ポリペプチドを含む不溶性画分を
得た。その画分に10mlの 9Ｍ尿素を含むバッファA を加
えて融合ポリペプチドを可溶化抽出した。可溶化した抽
出物を S−Sepharose を用いたイオン交換カラムクロマ
トグラフィーに掛けてＮａＣｌの 0Ｍから 0.5Ｍまでの
濃度勾配により融合ポリペプチドの精製を行った。(Ii) Expression and Purification of Polypeptide Encoded by Clone C11-7 Escherichia coli HB101 Having Expression Plasmids Trp / TrpE / C11-7
3 ml of 2YT medium containing 50 μg / ml of ampicillin
Inoculate (1.6% tryptone, 1% yeast extract, 0.5% NaCl) and incubate at 37 ℃ for 9 hours. 50 μg of 1 ml of this culture
100 ml of M9-CA medium (0.6% N containing ampicillin / ml)
a 2 HPO 4 , 0.5% KH 2 PO 4 , 0.5% NaC
1, 0.1% NH 4 Cl, 0.1 mM CaCl 2 , 2 mM Mg
The cells were subcultured in SO 4 , 0.5% casamino acid, 0.2% glucose) and cultured at 37 ° C. for 21 hours. In addition, 18 ml of the main culture solution
It was subcultured in 1.2 l of M9-CA medium and cultured at 37 ° C. OD
Indole acrylic acid was added to a final concentration of 40 mg / l at 600 = 0.3, and the cells were further cultured for 16 hours. The culture was centrifuged to collect bacterial cells. 20 ml buffer on the cells
A (50 mM Tris-HCl (pH8.0), 1 mM EDTA, 30 mM NaCl) was added and suspended, and centrifugation was carried out again to obtain 2.6 g of the expressing bacterial cells. The obtained bacterial cells were suspended in 10 ml of buffer A, the E. coli membrane was disrupted by ultrasonic disruption, and then centrifuged,
An insoluble fraction containing a fusion polypeptide of the polypeptide encoded by the non-A non-B hepatitis virus cDNA and TrpE was obtained. The fusion polypeptide was solubilized and extracted by adding 10 ml of buffer A containing 9 M urea to the fraction. The solubilized extract was subjected to ion exchange column chromatography using S-Sepharose to purify the fusion polypeptide by a concentration gradient of NaCl from 0M to 0.5M.
【００６９】実施例５非Ａ非Ｂ型肝炎患者血清中の抗非Ａ非Ｂ型肝炎ウイルス
抗体の測定 （ｉ）ウェスタンブロット法による測定 実施例４により精製された発現産物を SDS−ポリアクリ
ルアミドゲル電気泳動［Laemmli,Nature 277,680(197
0)］を行った後、常法に従ってニトロセルロースフィル
ター（Bio-rad,Trans-blot）にブロッティングを行っ
た。このフィルターに対して正常人血清および非Ａ非Ｂ
型肝炎患者血清をそれぞれ２検体ずつウエスタンブロッ
ティングを行い精製抗原との反応を確認した。第２図に
示したように正常人血清に対しては全く反応していない
が、患者血清に対しては２例とも強く反応した。Example 5 Anti-non-A non-B hepatitis virus in serum of non-A non-B hepatitis patients
Measurement of antibody (i) Measurement by Western blotting The expression product purified in Example 4 was subjected to SDS-polyacrylamide gel electrophoresis [Laemmli, Nature 277,680 (197).
0)] and then blotting was performed on a nitrocellulose filter (Bio-rad, Trans-blot) according to a conventional method. Normal human serum and non-A non-B against this filter
Western blotting was performed on two hepatitis patient sera, and the reaction with the purified antigen was confirmed. As shown in FIG. 2, it did not react at all with normal human serum, but strongly reacted with patient serum in both cases.
【００７０】（ii）酵素抗体測定法（ELISA 法）による
測定 非Ａ非Ｂ型肝炎患者の血清または血液中の特異抗体を測
定する手段の一つとしてELISA 法が使用できる。方法は
常法を用いて行い、先ず 5μｇ／mlの精製抗原をマイク
ロプレートに固定し、希釈被検血清を加えてインキュベ
ートする。洗浄後酵素標識した抗ヒト免疫グロブリンと
ともにインキュベートし、洗浄後基質を添加し、酵素活
性を比色法で測定し、抗原と反応した抗体量の測定を行
った。結果を表１に示す。現在上市されているカイロン
社製のキット（オーソHCV Ab ELISAテスト）と陽性率を
比較したところ、カイロン社のものは78％であるのに対
して、本発明による発現抗原を用いた場合は91％の陽性
率であった。なお表中、ＨＣＶ遺伝子／ＰＣＲは比較例
として第１群のプライマーを用いた遺伝子増幅法により
血清中のウイルス遺伝子の存在を調べたものであり、Ｐ
ＣＲの結果と本発明による発現抗原を用いたELISA の結
果はよく一致している。(Ii) By enzyme-linked immunosorbent assay (ELISA)
Measurement ELISA can be used as one of the means to measure the specific antibody in serum or blood of non-A non-B hepatitis patients. The method is carried out by a conventional method. First, 5 μg / ml of purified antigen is immobilized on a microplate, and diluted test serum is added and incubated. After washing, the plate was incubated with an enzyme-labeled anti-human immunoglobulin, after washing, a substrate was added, the enzyme activity was measured by a colorimetric method, and the amount of antibody that reacted with the antigen was measured. The results are shown in Table 1. When the positive rate is compared with the kit (Ortho HCV Ab ELISA test) manufactured by Chiron currently on the market, 78% is obtained by Chiron, whereas it is 91 when the expressed antigen according to the present invention is used. The positive rate was%. In the table, HCV gene / PCR was obtained by examining the presence of viral genes in serum by the gene amplification method using the first group of primers as a comparative example.
The result of CR and the result of ELISA using the expressed antigen according to the present invention are in good agreement.
【００７１】[0071]
【表１】 実施例６非Ａ非Ｂ型患者血漿中の非Ａ非Ｂ型肝炎ウイルス遺伝子
第２群のＲＴ−ＰＣＲ法による検出 得られたｃＤＮＡクローンは、核酸レベル、アミノ酸レ
ベルでのホモロジー比較で、第１群、第２群に分類でき
ることが分った。非Ａ非Ｂ型肝炎患者の血漿中の非Ａ非
Ｂ型肝炎ウイルス遺伝子を検出する方法としてＲＴ−Ｐ
ＣＲ法が考えられる。[Table 1] Example 6 Non-A non-B hepatitis virus genes in plasma of non-A non-B patients
Detection of the second group by the RT-PCR method The obtained cDNA clones were found to be classified into the first group and the second group by the homology comparison at the nucleic acid level and the amino acid level. RT-P as a method for detecting non-A non-B hepatitis virus gene in plasma of non-A non-B hepatitis patient
CR method is considered.
【００７２】第１群の非Ａ非Ｂ型肝炎ウイルス遺伝子を
検出するためには、カイロン社由来のＨＣＶの塩基配
列、又は宮村ら（前掲）のＪ１の塩基配列よりプライマ
ーを合成し、ＰＣＲ法を行うことが可能である。しか
し、第２群の非Ａ非Ｂ型肝炎ウイルス遺伝子を検出する
ためには、第２群のクローンの塩基配列より各クローン
間でよく保存されている領域をプライマーとして使うこ
とが望ましく、さらに効率よりＤＮＡ断片を増幅できる
プライマーを検討することが大切である。第２群のＨＣ
Ｖを検出するため、以下の方法でＲＴ−ＰＣＲを行っ
た。In order to detect the non-A non-B hepatitis virus gene of the first group, a primer was synthesized from the nucleotide sequence of HCV derived from Chiron or the nucleotide sequence of J1 of Miyamura et al. It is possible to However, in order to detect the non-A non-B hepatitis virus gene of the second group, it is desirable to use a region well conserved among the clones as a primer based on the nucleotide sequence of the clones of the second group. It is important to consider primers that can amplify more DNA fragments. HC of the second group
In order to detect V, RT-PCR was performed by the following method.
【００７３】非Ａ非Ｂ型肝炎患者血漿１００μｌに６Ｍ
のＧＴＣ液（６Ｍグアニジンチオシアネート、２５ｍＭ
クエン酸ナトリウム、０．５％サルコシル、０．２Ｍメ
ルカプトエタノール）３００μｌを加え撹拌する。さら
に４０μｌの２Ｍ酢酸ナトリウム（ｐＨ５．２）、４０
０μｌフェノール、８０μｌクロロホルム／イソアミル
アルコール（４９：１）を加えよく撹拌する。水溶液層
をとりイソプロピルアルコールを加え、これを遠心し、
沈澱を得る。これをＲＮＡとしてｃＤＮＡ合成を行っ
た。ｃＤＮＡ合成はTris−HCl 10mM、ゼラチン0.01％、
dNTP 各１mM、MgCl2 4mM, DTT 1mM、プライマー100 pm
ole の反応液にRNaseインヒビター、逆転写酵素を加
え、３７℃２時間反応して行った。このｃＤＮＡを用い
ＰＣＲを行った。ＰＣＲはバンドを検出するための感度
と特異性を上げるために、２ステップ法を用いた。すな
わち２種のプライマーで１回目のＰＣＲをかける（lst
step PCR）。次にそのＰＣＲ産物の内側に存在するプラ
イマー２種を用い２回目のＰＣＲをかける（2nd step P
CR）方法である。ＰＣＲの条件は、反応液 100μｌにcD
NA、Tris-HCl 10mM ，ゼラチン0.01％ dNTP 各２mM, Mg
Cl2 1.5mM，プライマー各50pmole を加え、アンプリフ
ィケーションサイクルは変性９４℃、１．５分、アニー
リング５０℃、２分、伸長７０℃、２分の条件で３５サ
イクル行った。いくつかのプライマーを検討した結果以
下の４種のプライマーを用いると第２群に特異的なＤＮ
Ａ断片が検出可能となった。6M in 100 μl of plasma of non-A non-B hepatitis patient
GTC solution (6M guanidine thiocyanate, 25 mM
300 μl of sodium citrate, 0.5% sarcosyl, 0.2M mercaptoethanol) is added and stirred. An additional 40 μl of 2M sodium acetate (pH 5.2), 40
Add 0 μl phenol and 80 μl chloroform / isoamyl alcohol (49: 1) and stir well. Take the aqueous layer, add isopropyl alcohol, centrifuge it,
Get a precipitate. This was used as RNA for cDNA synthesis. cDNA synthesis is Tris-HCl 10 mM, gelatin 0.01%,
dNTP 1 mM each, MgCl 2 4 mM, DTT 1 mM, primer 100 pm
RNase inhibitor and reverse transcriptase were added to the reaction solution of ole, and the reaction was carried out at 37 ° C. for 2 hours. PCR was performed using this cDNA. PCR used a two-step method to increase the sensitivity and specificity for detecting bands. That is, the first PCR is applied with two kinds of primers (lst
step PCR). Next, a second PCR is applied using the two primers present inside the PCR product (2nd step P
CR) method. PCR conditions are 100 μl of reaction solution and cD
NA, Tris-HCl 10 mM, gelatin 0.01% dNTP 2 mM each, Mg
Cl 2 1.5 mM and 50 pmole of each primer were added, and the amplification cycle was 35 cycles of denaturation 94 ° C., 1.5 minutes, annealing 50 ° C., 2 minutes, extension 70 ° C., 2 minutes. As a result of investigating several primers, DNs specific to the second group were obtained when the following four kinds of primers were used.
The A fragment became detectable.
【００７４】 ｌｓｔ ｓｔｅｐ ＰＣＲ ｋｋ２１：５′−ＧＧＡＴＡＣＡＣＣＧＧＴＧＡＣＴＴＴＧＡ−３′ ｋｋ２２：５′−ＴＧＣＡＴＧＣＡＣＧＴＧＧＣＧＡＴＧＴＡ−３′ ２ｎｄ ｓｔｅｐ ＰＣＲ ｋｋ２６：５′−ＧＡＴＧＣＣＣＡＣＴＴＣＣＴＣＴＣＣＣＡ−３′ ｋｋ２７：５′−ＧＴＣＡＧＧＧＴＡＡＣＣＴＣＧＴＴＧＧＴ−３′ この４種のプライマーを用いたＰＣＲで２０６塩基対の
ＤＮＡ断片が検出される。コントロールとして宮村ら
（前掲）のＪ１の塩基配列よりプライマーを合成し、第
１群のＤＮＡ断片の検出を行った。表２に非Ａ非Ｂ型肝
炎患者の血漿からのＰＣＲの結果を示した。1st step PCR kk21: 5′-GGATACACCGGGTGAACTTTGA-3 ′ kk22: 5′-TGCATGCACGTTGGCGATGTA-3 ′ 2nd step PCR kk26: 5′-GATGCCCACTTCCTCTCCCA-3G-TGCGCTC5G-TACGTCC3G-GTCACTCACAG′GACTACTGCAG: A DNA fragment of 206 base pairs is detected by PCR using. As a control, a primer was synthesized from the J1 nucleotide sequence of Miyamura et al. (Supra), and the first group of DNA fragments was detected. Table 2 shows the results of PCR from plasma of non-A non-B hepatitis patients.
【００７５】[0075]
【表２】 さらに、第１群のプライマーを用いるＰＣＲ（１群ＰＣ
Ｒ）、第２群のプライマーを用いるＰＣＲ（２群ＰＣ
Ｒ）の両者によってＤＮＡ断片を検出することができ、
第１群および第２群両方に関連するウイルス遺伝子をも
つと考えられる検体No.3、No.5についてシークエンスを
行った。表３は第２群ＰＣＲで得られたＤＮＡ断片の核
酸配列を第２群のクローンであるC10-13と比較したもの
であるが、各々８５％と８８％のホモロジーを示し、第
２群を検出しているものと考えられる。この２つの核酸
配列を第１群である宮村ら（前掲）のＪ１と比較すると
６４．８％と６８％のホモロジーしか示さなかった。こ
のホモロジー検索により２群ＰＣＲに用いたプライマー
は特異的に第２群のウイルス遺伝子を検出できると考え
られる。[Table 2] In addition, PCR using the first group of primers (1st group PC
R), PCR using the second group of primers (2 group PC
R) can detect a DNA fragment,
The samples No. 3 and No. 5, which are considered to have viral genes related to both the first group and the second group, were sequenced. Table 3 compares the nucleic acid sequences of the DNA fragments obtained by the second group PCR with the clone of the second group, C10-13, and shows a homology of 85% and 88%, respectively. It is considered to have been detected. Comparing these two nucleic acid sequences with J1 of the first group, Miyamura et al. (Supra), it showed only 64.8% and 68% homology. By this homology search, it is considered that the primers used for the second group PCR can specifically detect the second group viral genes.
【００７６】[0076]
【表３】 [Table 3]
【００７７】[0077]
【発明の効果】本発明によって得られたcDNA配列は、非
Ａ非Ｂ型肝炎に特異的であり、これらの遺伝子を微生
物、例えば大腸菌等を利用した蛋白質発原系に組み込む
ことによって産生されたポリペプチドは、多数の非Ａ非
Ｂ型肝炎患者の血清と反応するため、極めて感度のよい
診断キットを作製することができる。また、これらを直
接プローブとして診断に用いることも可能である。更
に、これらの配列に基づいて特異性の高いプローブを合
成し、必要ならば遺伝子増幅法（ＰＣＲ）と組み合わせ
ることにより、効率よく非Ａ非Ｂ型肝炎に特異的な遺伝
子を得ることもできる。The cDNA sequence obtained by the present invention is specific to non-A non-B hepatitis, and was produced by incorporating these genes into a protein origin system utilizing microorganisms such as Escherichia coli. Since the polypeptide reacts with the sera of many non-A non-B hepatitis patients, an extremely sensitive diagnostic kit can be made. It is also possible to directly use these as probes for diagnosis. Furthermore, by synthesizing a highly specific probe based on these sequences and, if necessary, combining it with a gene amplification method (PCR), a gene specific to non-A non-B hepatitis can be efficiently obtained.
【００７８】また、これらのポリペプチドは、非Ａ非Ｂ
型肝炎ウイルス抗原としての機能を有するため、ワクチ
ンとしてこのポリペプチドを免疫することにより非Ａ非
Ｂ型肝炎ウイルスの感染を防御できることも期待でき
る。In addition, these polypeptides are non-A non-B
Since it has a function as a hepatitis B virus antigen, it can be expected that immunization with this polypeptide as a vaccine can protect against infection with non-A non-B hepatitis virus.
【００７９】更にまた、これらのポリペプチドをウサギ
等に免疫することにより、患者体内のウイルス検出、分
離に有用な抗体を得ることができるだろう。Furthermore, by immunizing rabbits and the like with these polypeptides, it will be possible to obtain antibodies useful for detecting and isolating the virus in the patient's body.
【００８０】[0080]
配列番号：１ 配列の長さ：763 塩基対 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 genomic RNA 配列 CG CAG TCA TTC CAA GTG GCC CAT CTA CAC GCT CCC ACT GGC AGC GGC 47 Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly 1 5 10 15 AAG AGT ACT AAA GTG CCG GCT GCA TAT GCC AGC CAA GGG TAC AAG GTG 95 Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ser Gln Gly Tyr Lys Val 20 25 30 CTC GTC CTC AAC CCG TCC GTT GCC GCC ACC TTA GGT TTT GGA GCG TAT 143 Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr 35 40 45 ATG TCT AAG GCA CAT GGC ACC GAC CCC AAC ATC AGA ACT GGG GTA AGG 191 Met Ser Lys Ala His Gly Thr Asp Pro Asn Ile Arg Thr Gly Val Arg 50 55 60 ACT ATC ACC ACA GGC GCC CCC ATC ACG TAC TCC ACC TAC GGC AAG TTC 239 Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe 65 70 75 CTT GCC GAC GGT GGT TGT TCT GGG GGC GCT TAT GAC ATC ATA ATG TGT 287 Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Met Cys 80 85 90 95 GAT GAG TGC CAC TCA ACT GAC GCG ACT TCC ATC TTG GGC ATC GGC ACG 335 Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr 100 105 110 GTC CTG GAC CAA GCG GAG ACG GCT GGA GCA CGG CTC GTC GTG CTC GCC 383 Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala 115 120 125 ACC GCT ACG CCT CCG GGA TCG GTC ACC GTG CCA CAC CCG AAT ATT GAG 431 Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu 130 135 140 GAG GTG GCC CTG TCT AAC ACT GGA GAG ATC CCC TTC TAT GGC AAA GGC 479 Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Gly 145 150 155 ATC CCC ATT GAA GTC ATC AAG GGG GGA AGG CAT CTC ATT TTC TGC CAT 527 Ile Pro Ile Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His 160 165 170 175 TCC AAG AAG AAG TGC GAC GAG CTC GCC GCG AAG TTG TCA GGC CTC GGG 575 Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly 180 185 190 ATT AAT GCT GTG GCA TAC TAC CGG GGT CTT GAT GTG TCC GTC ATA CCG 623 Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro 195 200 205 ACC AGC GGA GAC GTC GTT GTC GTG GCA ACA GAC GCT CTA ATG ACG GGC 671 Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly 210 215 220 TAT ACC GGC GAT TTT GAC TCA GTG ATC GAC TGT AAC ACA TGC GTC ACC 719 Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr 225 230 235 CAG ACA GTC GAC TTC AGC TTG GAC CCC ACC TTC ACC ATT GAG AC 763 Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu 240 245 250 配列番号：２ 配列の長さ：615 塩基対 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 genomic RNA 配列 C ACG CCC GGT TTG CCC GTG TGT CAA GAC CAC CTG GAG TTC TGG GAA GCG 49 Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala 1 5 10 15 GTC TTC ACA GGT CTC ACG CAC ATT GAT GCC CAC TTC CTC TCC CAG ACA 97 Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr 20 25 30 AAG CAA GGA GGA GAC AAC TTC GCG TAT CTA ACG GCC TAC CAG GCC ACA 145 Lys Gln Gly Gly Asp Asn Phe Ala Tyr Leu Thr Ala Tyr Gln Ala Thr 35 40 45 GTG TGC GCT AGG GCA AAG GCC CCT CCT CCC TCG TGG GAT GTG ATG TGG 193 Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Val Met Trp 50 55 60 AAA TGT CTA GCT AGG CTG AAG CCT ACA CTA ATT GGT CCT ACC CCC CTC 241 Lys Cys Leu Ala Arg Leu Lys Pro Thr Leu Ile Gly Pro Thr Pro Leu 65 70 75 80 CTG TAC CGC TTG GGT GCC GTG ACC AAC GAG GTT ACC CTG ACG CAC CCC 289 Leu Tyr Arg Leu Gly Ala Val Thr Asn Glu Val Thr Leu Thr His Pro 85 90 95 GTG ACG AAA TAC ATC GCC ACG TGC ATG CAA GCT GAC CTC GAG ATC ATG 337 Val Thr Lys Tyr Ile Ala Thr Cys Met Gln Ala Asp Leu Glu Ile Met 100 105 110 ACG AGC ACA TGG GTC CTA GCA GGG GGG GTG CTA GCC GCC GTG GCA GCT 385 Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu Ala Ala Val Ala Ala 115 120 125 TAC TGC CTG GCA ACC GGC TGT GTT TCC ATC ATC GGC CGC CTA CAC CTG 433 Tyr Cys Leu Ala Thr Gly Cys Val Ser Ile Ile Gly Arg Leu His Leu 130 135 140 AAT GAT CAA GTG GTT GTG ACT CCT GAC AAA GAA ATC TTA TAT GAG GCC 481 Asn Asp Gln Val Val Val Thr Pro Asp Lys Glu Ile Leu Tyr Glu Ala 145 150 155 160 TTT GAT GAG ATG GAA GAA TGC GCC TCC AAA GCC GCC CTC ATT GAG GAA 529 Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Ala Ala Leu Ile Glu Glu 165 170 175 GGG CAG CGG ATG GCG GAG ATG CTC AAG TCT AAG ATA CAA GGC CTC CTA 577 Gly Gln Arg Met Ala Glu Met Leu Lys Ser Lys Ile Gln Gly Leu Leu 180 185 190 CAA CAG GCC ACA AGA CAG GCC CAA GAC ATA CAG CCA GC 615 Gln Gln Ala Thr Arg Gln Ala Gln Asp Ile Gln Pro 195 200 配列番号：３ 配列の長さ：771 塩基対 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 genomic RNA 配列 GT GAG CGA GCC TCA GGA ATG TTT GAC AGT GTA GTG CTC TGT GAG TGC 47 Glu Arg Ala Ser Gly Met Phe Asp Ser Val Val Leu Cys Glu Cys 1 5 10 15 TAT GAC GCA GGG GCT GCA TGG TAC GAG CTT ACA CCA GCG GAG ACC ACC 95 Tyr Asp Ala Gly Ala Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr 20 25 30 GTC AGG CTC AGA GCG TAT TTC AAC ACA CCT GGC TTG CCT GTG TGT CAA 143 Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly Leu Pro Val Cys Gln 35 40 45 GAC CAT CTT GAG TTC TGG GAG GCA GTT TTC ACC GGC CTC ACA CAC ATA 191 Asp His Leu Glu Phe Trp Glu Ala Val Phe Thr Gly Leu Thr His Ile 50 55 60 GAT GCC CAC TTC CTT TCC CAG ACA AAG CAA GCA GGG GAC AAT TTC GCA 239 Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Ala 65 70 75 TAC TTG ACA GCC TAC CAG GCT ACA GTG TGC GCC AGA GCC AAA GCC CCT 287 Tyr Leu Thr Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala Pro 80 85 90 95 CCC CCG TCC TGG GAC GTC ATG TGG AAG TGC CTG ACT CGG CTC AAG CCC 335 Pro Pro Ser Trp Asp Val Met Trp Lys Cys Leu Thr Arg Leu Lys Pro 100 105 110 ACG CTT GTG GCC CCT ACA CCC CTT CTG TAC CGT TTA GGC TCT GTT ACT 383 Thr Leu Val Ala Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ser Val Thr 115 120 125 AAC GAG GTC ACC CTC ACA CAT CCT GTG ACG AAA TAC ATC GCC ACT TGC 431 Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Ala Thr Cys 130 135 140 ATG CAA GCT GAC CTT GAG GTC ATG ACC AGC ACG TGG GTC CTA GCT GGG 479 Met Gln Ala Asp Leu Glu Val Met Thr Ser Thr Trp Val Leu Ala Gly 145 150 155 GGG GTC TTG GCA GCC GTC GCC GCG TAT TGC CTG GCG ACT GGG TGT GTC 527 Gly Val Leu Ala Ala Val Ala Ala Tyr Cys Leu Ala Thr Gly Cys Val 160 165 170 175 TCC ATC ATC GGC CGC TTG CAC ATC AAT CAG CGA GCC GTC GTT GCA CCA 575 Ser Ile Ile Gly Arg Leu His Ile Asn Gln Arg Ala Val Val Ala Pro 180 185 190 GAC AAG GAG GTC CTT TAT GAG GCT TTT GAT GAG ATG GAG GAG TGT GCC 623 Asp Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala 195 200 205 TCT AAA GCG GCT CTC ATT GAA GAG GGG CAG CGG ATA GCC GAG ATG CTG 671 Ser Lys Ala Ala Leu Ile Glu Glu Gly Gln Arg Ile Ala Glu Met Leu 210 215 220 AAG TCC AAG ATC CAA GGC TTA TTG CAG CAA GCC TCT AAA CAG GCC CAG 719 Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser Lys Gln Ala Gln 225 230 235 GAC ATA CAA CCC GCT GTG CAG CCT CAT GGC CCA AGG TGG AGC AAT TCT 767 Asp Ile Gln Pro Ala Val Gln Pro His Gly Pro Arg Trp Ser Asn Ser 240 245 250 255 GGG C 771 Gly 配列番号：４ 配列の長さ：630 塩基対 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 genomic RNA 配列 C TGG TAT GAA CTT ACG CCT GCT GAG ACT ACG GTG AGA CTC CGG GCC TAT 49 Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr 1 5 10 15 TTC AAC ACG CCC GGC CTG CCT GTG TGT CAA GAC CAC CTG GAA TTC TGG 97 Phe Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp 20 25 30 GAG GCG GTC TTC ACA GGT CTC ACA CAC ATC GAT GCC CAC TTC CTC TCC 145 Glu Ala Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser 35 40 45 CAG ACG AAG CAA GGA GGA GAT AAC TTT GCA TAT TTA ACA GCC TAC CAG 193 Gln Thr Lys Gln Gly Gly Asp Asn Phe Ala Tyr Leu Thr Ala Tyr Gln 50 55 60 GCC ACA GTC TGC GCT AGG GCA AAG GCT CCC CCT CCT TCG TGG GAC GTG 241 Ala Thr Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Val 65 70 75 80 ATG TGG AAG TGT TTG ATT AGG CTC AAA CCT ACA CTG ACT GGT CCT ACC 289 Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu Thr Gly Pro Thr 85 90 95 CCC CTC CTG TAC CGC TTG GGT GCC GTG ACC AAC GAG GTT ACC CTG ACT 337 Pro Leu Leu Tyr Arg Leu Gly Ala Val Thr Asn Glu Val Thr Leu Thr 100 105 110 CAC CCC ATG ACG AAA TAT ATC GCC ACT TGT ATG CAA GCT GAT CTT GAG 385 His Pro Met Thr Lys Tyr Ile Ala Thr Cys Met Gln Ala Asp Leu Glu 115 120 125 ATC ATG ACA AGC ACA TGG GTC TTG GCG GGG GGG GTG CTA GCC GCT GTG 433 Ile Met Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu Ala Ala Val 130 135 140 GCA GCT TAC TGC CTA GCG ACC GGC TGC ATT TCC ATC ATT GGC CGC CTT 481 Ala Ala Tyr Cys Leu Ala Thr Gly Cys Ile Ser Ile Ile Gly Arg Leu 145 150 155 160 CAC CTG AAT GAT CGG GTG GTC GTG ACC CCT GAT AAG GAA ATT TTA TAT 529 His Leu Asn Asp Arg Val Val Val Thr Pro Asp Lys Glu Ile Leu Tyr 165 170 175 GAG GCC TTT GAT GAG ATG GAA GAG TGC GCC TCC AAA GCC GCC CTC ATT 577 Glu Ala Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Ala Ala Leu Ile 180 185 190 GAG GAA GGG CAG CGG ATG GCG GAG ATG CTG AAG TCT AAA ATA CAA GGC 625 Glu Glu Gly Gln Arg Met Ala Glu Met Leu Lys Ser Lys Ile Gln Gly 195 200 205 CTC TT 630 Leu 配列番号：５ 配列の長さ：1426塩基対 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 genomic RNA 配列 GGG ATC AAC CCT AAC ATC AGG ACC GGA GTA CGG ACC GTG ACC ACC GGG 48 Gly Ile Asn Pro Asn Ile Arg Thr Gly Val Arg Thr Val Thr Thr Gly 1 5 10 15 GAC TCC ATC ACC TAC TCC ACT TAT GGC AAG TTT ATC GCA GAT GGA GGT 96 Asp Ser Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Ile Ala Asp Gly Gly 20 25 30 TGC GCA CAT GGT GCC TAT GAC GTC ATC ATA TGC GAC GAA TGC CAT TCA 144 Cys Ala His Gly Ala Tyr Asp Val Ile Ile Cys Asp Glu Cys His Ser 35 40 45 GTG GAC GCT ACT ACC ATC CTT GGC ATT GGA ACA GTC CTT GAC CAG GCT 192 Val Asp Ala Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala 50 55 60 GAG ACC GCA GGT GCC AGG CTA GTG GTT TTA GCC ACA GCC ACG CCA CCC 240 Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro 65 70 75 80 GGT ACG GTA ACA ACT CCC CAC GCT AAC ATA GAG GAG GTG GCC CTT GGT 288 Gly Thr Val Thr Thr Pro His Ala Asn Ile Glu Glu Val Ala Leu Gly 85 90 95 CAC GAA GGC GAG ATT CCT TTT TAT GGC AAG GCT ATT CCC CTA GCT TTC 336 His Glu Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Ala Phe 100 105 110 ATC AAG GGG GGC AGA CAC CTA ATT TTT TGC CAT TCA AAG AAG AAG TGC 384 Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys 115 120 125 GAC GAG CTC GCA GCA GCC CTT CGG GGC ATG GGT ATC AAT GCC GTT GCC 432 Asp Glu Leu Ala Ala Ala Leu Arg Gly Met Gly Ile Asn Ala Val Ala 130 135 140 TAC TAC AGG GGT CTC GAC GTC TCC GTT ATA CCA ACT CAA GGA GAC GTG 480 Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Gln Gly Asp Val 145 150 155 160 GTG GTT GTC GCC ACC GAT GCC CTA ATG ACT GGA TAC ACC GGT GAC TTT 528 Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe 165 170 175 GAC TCT GTC ATC GAC TGC AAC GTT GCA GTC ACT CAG ATT GTT GAC TTT 576 Asp Ser Val Ile Asp Cys Asn Val Ala Val Thr Gln Ile Val Asp Phe 180 185 190 AGC CTA GAC CCA ACT TTT ACC ATC ACC ACT CAA ACC GTC CCT CAG GAG 624 Ser Leu Asp Pro Thr Phe Thr Ile Thr Thr Gln Thr Val Pro Gln Glu 195 200 205 GCT GTC TCC CGT AGT CAA CGT AGA GGG AGA ACT GGG AGG GGG CGA CTG 672 Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Leu 210 215 220 GGC ACT TAC AGG TAT GTC TCG TCA GGC GAG AGG CCG TCT GGG ATG TTC 720 Gly Thr Tyr Arg Tyr Val Ser Ser Gly Glu Arg Pro Ser Gly Met Phe 225 230 235 240 GAC AGC GTA GTA CTC TGC GAG TGC TAT GAT GCC GGG GCA GCC TGG TAC 768 Asp Ser Val Val Leu Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr 245 250 255 GAG CTT ACA CCT GCT GAG ACC ACA GTG AGA CTC CGG GCT TAT TTC AAC 816 Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Phe Asn 260 265 270 ACG CCC GGT TTG CCC GTG TGT CAA GAC CAC CTG GAG TTC TGG GAA GCG 864 Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala 275 280 285 GTC TTC ACA GGT CTC ACG CAC ATT GAT GCC CAC TTC CTC TCC CAG ACA 912 Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr 290 295 300 AAG CAA GGA GGA GAC AAC TTC GCG TAT CTA ACG GCC TAC CAG GCC ACA 960 Lys Gln Gly Gly Asp Asn Phe Ala Tyr Leu Thr Ala Tyr Gln Ala Thr 305 310 315 320 GTG TGC GCT AGG GCA AAG GCC CCT CCT CCC TCG TGG GAT GTG ATG TGG 1008 Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Val Met Trp 325 330 335 AAA TGT CTA GCT AGG CTG AAG CCT ACA CTA ATT GGT CCT ACC CCC CTC 1056 Lys Cys Leu Ala Arg Leu Lys Pro Thr Leu Ile Gly Pro Thr Pro Leu 340 345 350 CTG TAC CGC TTG GGT GCC GTG ACC AAC GAG GTT ACC CTG ACG CAC CCC 1104 Leu Tyr Arg Leu Gly Ala Val Thr Asn Glu Val Thr Leu Thr His Pro 355 360 365 GTG ACG AAA TAC ATC GCC ACG TGC ATG CAA GTG AAC CTC GAG ATC ATG 1152 Val Thr Lys Tyr Ile Ala Thr Cys Met Gln Val Asn Leu Glu Ile Met 370 375 380 ACG AGC ACA TGG GTC CTA GCA GGG GGG GTG CTA GCC GCC GTG GCA GCT 1200 Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu Ala Ala Val Ala Ala 385 390 395 400 TAC TGC CTG GCA ACC GGC TGT GTT TCC ATC ATC GGC CGC CTA CAC CTG 1248 Tyr Cys Leu Ala Thr Gly Cys Val Ser Ile Ile Gly Arg Leu His Leu 405 410 415 AAT GAT CAA GTG GTT GTG ACT CCT GAC AAA GAA ATC TTA TAT GAG GCC 1296 Asn Asp Gln Val Val Val Thr Pro Asp Lys Glu Ile Leu Tyr Glu Ala 420 425 430 TTT GAT GAG ATG GAA GAA TGC GCC TCC AAA GCC GCC CTC ATT GAG GAA 1344 Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Ala Ala Leu Ile Glu Glu 435 440 445 GGG CAG CGG ATG GCG GAG ATG CTC AAG TCT AAG ATA CAA GGC CTC CTA 1392 Gly Gln Arg Met Ala Glu Met Leu Lys Ser Lys Ile Gln Gly Leu Leu 450 455 460 CAA CAG GCC ACA AGA CAG GCC CAA GAC ATA CAG C 1426 Gln Gln Ala Thr Arg Gln Ala Gln Asp Ile Gln 465 470 475 配列番号：６ 配列の長さ：855 塩基対 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 genomic RNA 配列 CG CAG ACA TTC CAA GTG GCC CAT CTG CAC GCT CCC ACT GGT AGC GGC 47 Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly 1 5 10 15 AAG AGC ACT AAG GTG CCG GCT GCA TAT GCG GCC CAA GGG TAC AAG GTA 95 Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val 20 25 30 CTC GTC CTG AAC CCG TCC GTT GCC GCC ACT TTA GCC TTT GGG GCG TAC 143 Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Ala Phe Gly Ala Tyr 35 40 45 ATG TCT AAG GCA CAT GGT GTC GAC CCT AAC ATC AGA ACT GGG GTG AGG 191 Met Ser Lys Ala His Gly Val Asp Pro Asn Ile Arg Thr Gly Val Arg 50 55 60 ACC ATC ACC ACG GGC GCT CCC ATC ACG TAC TCC ACC TAT GGT AAG TTC 239 Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe 65 70 75 CTT GCC GAC GGT GGT TGC TCT GGG GGC GCC TAT GAC ATC ATA ATA TGT 287 Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys 80 85 90 95 GAT GAG TGC CAC TCA ACT GAC TCG ACA TCC ATC TTG GGC ATC GGC ACA 335 Asp Glu Cys His Ser Thr Asp Ser Thr Ser Ile Leu Gly Ile Gly Thr 100 105 110 GTC CTG GAC CAA GCG GAG ACG GCT GGA GCG CGG CTC GTC GTG CTC GCT 383 Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala 115 120 125 ACC GCT ACG CCT CCG GGA TCG GTC ACC GTG CCA CAT CCC AAT ATC GAG 431 Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu 130 135 140 GAG GTG GCC CTG TCC ACC ACT GGA GAG ATT CCC TTC TAC GGC AAA GCT 479 Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala 145 150 155 ATC CCC ATC GAG ACA ATC AAG GGG GGG AGG CAT CTC ATC TTC TGC CGT 527 Ile Pro Ile Glu Thr Ile Lys Gly Gly Arg His Leu Ile Phe Cys Arg 160 165 170 175 TCC AAG AAG AAG TGT GAC GAG CTC GCT GGA AAG CTG TCA GCC CTC GGA 575 Ser Lys Lys Lys Cys Asp Glu Leu Ala Gly Lys Leu Ser Ala Leu Gly 180 185 190 ATC AAC GCT GTA GCG TAC TAC CGG GGT CTT GAT GTA TCC GTC ATA CCG 623 Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro 195 200 205 ACC AGC GGA GAC GTC GTT GTC GTG GCA ACA GAC GCT CTA ATG ACG GGC 671 Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly 210 215 220 TAC ACC GGT GAC TTT GAT TCA GTG ATC GAC TGC AAT ACA TGT GTC ACC 719 Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr 225 230 235 CAG ACA GTC GAC TTC AGC TTG GAC CCT ACC TTC ACC ATT GAG ACG ACG 767 Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr 240 245 250 255 ACC GTG CCT CAA GAC GCG GTG TCA CGC TCG CAG CGG CGA GGC AGA ACT 815 Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr 260 265 270 GGT AGG GGT AGA GGG GGC ATA TAC AGG TTT GTG ACT CCA G 855 Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val Thr Pro 275 280 配列番号：７ 配列の長さ：315 塩基対 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 genomic RNA 配列 GAC GAG CTC GCC GCA AAG CTG TCA GGC CTC GGA GTC AAT GCT GTG GCA 48 Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Val Asn Ala Val Ala 1 5 10 15 TAC TAC CGG GGT CTC GAT GTG TCT GTC ATA CCG ACG AGC GGG GAC GTC 96 Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val 20 25 30 GTT GTT GTG GCA ACA GAC GCT CTA ATG ACG GGC TAT ACC GGC GAC TTT 144 Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe 35 40 45 GAC TCG GTG ATC GAC TGC AAT ACA TGT GTC ACC CAA ACA GTC GAT TTC 192 Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe 50 55 60 AGC TTG GAC CCT ACT TTC ACC ATT GAG ACG ACG ACC GTG CCC CAA GAC 240 Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln Asp 65 70 75 80 GCG GTG TCG CGC TCG CAG CGG CGA GGC AGG ACT GGT AGG GGC AGG GTG 288 Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Val 85 90 95 GGC ATA TAC AGG TTT GTG ACT CCC GAG 315 Gly Ile Tyr Arg Phe Val Thr Pro Glu 100 105 配列番号：８ 配列の長さ：911 塩基対 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 genomic RNA 配列 GT GAT GAG CTC GCC GCA AAG CTC TCA AGC CTC GGA CTC AAC GCT GTA 47 Asp Glu Leu Ala Ala Lys Leu Ser Ser Leu Gly Leu Asn Ala Val 1 5 10 15 GCA TAT TAC CGG GGT CTT GAT GTG TCC GTC ATA CCG ACT AGT GGA GAC 95 Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp 20 25 30 GTC GTT GTC GTG GCA ACA GAC GCT CTA ATG ACG GGC TAT ACC GGC GAC 143 Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp 35 40 45 TTT GAC TCA GTG ATC GAC TGT AAC ACA TGT GTC ACC CAG ACA GTT GAT 191 Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp 50 55 60 TTC AGC TTG GAT CCA ACC TTC ACC ATT GAG ACG ACG ACC GTG CCT CAA 239 Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln 65 70 75 GAC GCG GTG TCG CGC TCG CAG CGG CGA GGT AGG ACT GGC AGG GGC AGG 287 Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg 80 85 90 95 GGC GGC ATC TAT AGG TTT GTG ACT CCA GGA GAA CGG CCC TCG GGC ATG 335 Gly Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met 100 105 110 TTC GAT TCC TCG GTC CTG TGT GAG TGT TAT GAC GCG GGC TGT GCT TGG 383 Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp 115 120 125 TAT GAG CTC ACG CCC GCC GAG ACC ACG GTT AGG TTG CGG GCT TAC CTA 431 Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Leu 130 135 140 AAT ACA CCA GGG TTG CCC GTC TGC CAG GAC CAT CTG GAG TTC TGG GAG 479 Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu 145 150 155 GGC GTC TTC ACA GGC CTC ACC CAC ATA GAT GCC CAT TTC TTG TCT CAG 527 Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln 160 165 170 175 ACT AAG CAG GCA GGA CAC AAC TTT CCC TAC CTG GTG GCA TAC CAA GCT 575 Thr Lys Gln Ala Gly His Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala 180 185 190 ACA GTG TGC GCC AGG GCT CAG GCT CCA CCT CCA TCG TGG GAC CAA ATG 623 Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met 195 200 205 TGG AAG TGT CTC ATA CGG CTG AAA CCT ACG CTG CAC GGG CCA ACA CCC 671 Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro 210 215 220 CTG CTG TAT AGG CTA GGA GCC GTG GAA AAT GAG GTC ACC CTC ACA CAC 719 Leu Leu Tyr Arg Leu Gly Ala Val Glu Asn Glu Val Thr Leu Thr His 225 230 235 CCC ATA ACC AAA TTC ATC ATG GCA TGC ATG TCG GCT GAT CTG GAG GTC 767 Pro Ile Thr Lys Phe Ile Met Ala Cys Met Ser Ala Asp Leu Glu Val 240 245 250 255 GTC ACC AGC ACC TGG GTG CTG GTG GGC GGA GTC CTT GCA GCT CTG GCC 815 Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala 260 265 270 GCA TAT CGC CTG ACA ACA GGC AGC GTG GTC ATC GTG GGT AGG ATC ATC 863 Ala Tyr Arg Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile 275 280 285 TTG TCT GGG AGG CCG GCT GTC ATT CCC GAC AGG GAA GTC CTT TAC CGG 911 Leu Ser Gly Arg Pro Ala Val Ile Pro Asp Arg Glu Val Leu Tyr Arg 290 295 300 配列番号：９ 配列の長さ：489 塩基対 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 genomic RNA 配列 CG ACA ACC GTG CCC CAA GAC GCG GTG TCG CGC TCA CAA CGG CGG GGT 47 Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly 1 5 10 15 AGG ACA GGT AGG GGC AGG AGA GGC ATC TAC AGA TTT GTG ACT CCG GGA 95 Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly 20 25 30 GAA CGG CCC TCG GGC ATG TTC GAT TCT TCG GTC CTG TGT GAG TGC TAT 143 Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr 35 40 45 GAC GCG GGC TGC GCT TGG ATC GAG CTC ACG CCC GCC GAG ACC TCA GTT 191 Asp Ala Gly Cys Ala Trp Ile Glu Leu Thr Pro Ala Glu Thr Ser Val 50 55 60 AGG TTG CGG GCT TAC CTA AAT ACA CCA GGG TTG CCC GTC TGC CAG GAC 239 Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp 65 70 75 CAC CTG GAA TTC TGG GAG AGC GTC TTC ACA GGC CTC ACC CAT ATA GAT 287 His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp 80 85 90 95 GCC CAC TTC TTG TCC CAG ACC AAG CAG GCA GGA GAC AAC TTC CCC TAC 335 Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr 100 105 110 CTG GTA GCA TAC CAA GCT ACA GTG TGC GCC AGG GCC CAG GCT CCA CCA 383 Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro 115 120 125 CCA TCG TGG GAT CAA ATG TGG AAG TGT CTC ATA CGG CTG AAA CCT ACG 431 Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr 130 135 140 CTA CAC GGG CCA ACA CCC CTG TTG TAT AGG CTG GGA GCC GTC CAA AAT 479 Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn 145 150 155 GAG GTC ACC C 489 Glu Val Thr 160 配列番号：１０ 配列の長さ：1076塩基対 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 genomic RNA 配列 GT GGT CTC CTG GGT GCC ATC GTG GTC AGC CTA ACG GGC CGC GAC AAG 47 Gly Leu Leu Gly Ala Ile Val Val Ser Leu Thr Gly Arg Asp Lys 1 5 10 15 AAC CAG GTC GAG GGG GAG GTT CAG GTG GTC TCC ACC GCA ACG CAA TCT 95 Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser 20 25 30 TTC CTG GCG ACC TGC GTC AAT GGC GTG TGT TGG ACC GTC TAC CAT GGC 143 Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His Gly 35 40 45 GCC GGC TCG AAA ACC CTG GCC GGC CCG AAG GGT CCA GTC ACC CAA ATG 191 Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Val Thr Gln Met 50 55 60 TAC ACT AAT GTG GAC CAG GAC CTC GTC GGC TGG CCG GCG CCC TCC GGG 239 Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Ser Gly 65 70 75 GCG CGG TCC TTG ACA CCA TGC ACC TGC GGC AGC TCG GAC CTT TAC TTG 287 Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu 80 85 90 95 GTC ACG AGG CAT GCT GAT GTC ATT CCG GTG CGC CGG CGG GGC GAT AGC 335 Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser 100 105 110 AGG GGG AGC CTG CTT TCC CCC AGG CCC CTC TCC TAC TTG AAG GGC TCC 383 Arg Gly Ser Leu Leu Ser Pro Arg Pro Leu Ser Tyr Leu Lys Gly Ser 115 120 125 TCA GGT GGT CCA CTG CTT TGC CCC TCG GGG CAC ATT GTG GGC ATC TTC 431 Ser Gly Gly Pro Leu Leu Cys Pro Ser Gly His Ile Val Gly Ile Phe 130 135 140 CGG GCT GCC GTG TGC ACC CGG GGG GTT GCG AAG GCG GTG GAC TTT GTA 479 Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val 145 150 155 CCT GTC GAG TCT ATG GAA ACT ACT ATG CGG TCT CCG GTC TTC ACG GAT 527 Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp 160 165 170 175 AAT TCA TCC CCC CCG GCC GTA CCG CAG ACA TTC CAA GTG GCC CAT CTG 575 Asn Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu 180 185 190 CAT GCC CCC ACT GGC AGC GGC AAG AGC ACT AAG GTG CCG GCT GCA TAC 623 His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr 195 200 205 GCA GCC CAG GGA TAC AAG GTA CTC GTC CTG AAC CCG TCC GTT GCC GCC 671 Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala 210 215 220 ACC TTA GGT TTT GGA GCA TAT ATG TCC AAG GCA CAT GGT GTC GAC CCT 719 Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro 225 230 235 AAC ATC AGG ACT GGG GTA AGG ACC ATC ACT ACG GGC GCC CCC ATT ACA 767 Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr 240 245 250 255 TAC TCC ACC TAT GGC AAG TTT CTT GCC GAC GGT GGT TGC TCC GGG GGC 815 Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly 260 265 270 GCC TAT GAC ATC ATA ATA TGT GAT GAG TGC CAC TCA ACT GAC TCG ACT 863 Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr 275 280 285 TCC ATT TTG GGC ATT GGC ACG GTC CTG GAC CAA GCG GAG ACG GCT GGA 911 Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly 290 295 300 GCG CGG CTC GTC GTG CTC GCC ACC GCT ACG CCT CCA GGA TCG GTC ACT 959 Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr 305 310 315 GTG CCT CAT CCC AAC ATC GAG GAG GTG GCC TTG TCC AGC ACT GGA GAG 1007 Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Ser Thr Gly Glu 320 325 330 335 ATT CCC TTC TAT GGC AAA GCC ATC CCC ATT GAG ACC ATC AAG GGG GGA 1055 Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Thr Ile Lys Gly Gly 340 345 350 AGG CAT CTC ATT TTC TGC CAC 1076 Arg His Leu Ile Phe Cys His 355 配列番号：１１ 配列の長さ：284 塩基対 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 genomic RNA 配列 GTC GAC CCC AAT ATT AGA ACT GGG GTA AGG ACC ATC ACC ACG GGC GCT 48 Val Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala 1 5 10 15 CCC ATT ACG TAT TCT ACC TAT GGC AAA TTC CTT GCC GAC GGT GGT TGC 96 Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys 20 25 30 TCT GGG GGC GCC TAT GAC ATC ATA ATC TGT GAT GAG TGC CAC TCA ACT 144 Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr 35 40 45 GAC TCG ACT TCC ATC TTG GGT ATC GGC ACA GCC CTG GAC CAA GCG GAG 192 Asp Ser Thr Ser Ile Leu Gly Ile Gly Thr Ala Leu Asp Gln Ala Glu 50 55 60 ACG GCT GGA GCA CGG CTT GTC GTG CTC GCC ACC GCT ACG CCT CCA GGG 240 Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly 65 70 75 80 TCG GTC ACC GTG CCG CAT CCC AAC ATC GAG GAG GTA GCC TTG CC 284 Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu 85 90 配列番号：１２ 配列の長さ：641 塩基対 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 genomic RNA 配列 G GAC AAC TCA TCT CCC CCG GCG GTA CCG CAG ACA TTC CAG GTG GCC CAT 49 Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His 1 5 10 15 CTA CAC GCT CCC ACT GGC AGC GGC AAG AGC ACT AAG GTG CCG GCT GCA 97 Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala 20 25 30 TAT GCA GCC CAA GGG TAC AAA GTA CTC GTC CTG AAC CCG TCC GTT GCC 145 Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala 35 40 45 GCC ACC TTA AGT TTC GGG GCG TAT ATG TCC AAG GCA CAT GGT GTT GAC 193 Ala Thr Leu Ser Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp 50 55 60 CCT AAT ATC AGA ACT GGG ACA AGG ACC ATC ACC ACG GGC GCT CCC ATC 241 Pro Asn Ile Arg Thr Gly Thr Arg Thr Ile Thr Thr Gly Ala Pro Ile 65 70 75 80 ACG TAC TCC ACC TAT GGC AAG TTC CTT GCA GAC GGT GGT TGC TCC GGA 289 Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly 85 90 95 GGC GCC TAT GAC ATC ATA ATA TGC GAT GAG TGC CAC TCA ACA GAC TCG 337 Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser 100 105 110 ACT TCC ATC TTA GGC ATT GGT ACG GTC CTG GAC CAA GCG GAG ACG GCT 385 Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala 115 120 125 GGA GCG CGA CTC GTC GTG CTC GCC ACC GCT ACG CCT CCA GGA TCG GTC 433 Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val 130 135 140 ACT GTG CCA CAT CCC AAC ATC GAG GAG GTG GCC CTG TCC AAC ACT GGA 481 Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly 145 150 155 160 GAG ATT CCC TTC TAT GGC AAA GCC ATC CCC ATT GAG GCC ATC AAG GGG 529 Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Ala Ile Lys Gly 165 170 175 GGG AGG CAT CTC ATT TTC TGC CAT TCT AAG AAG AAG TGT GAT GAG CTC 577 Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu 180 185 190 GCC ACG AAG CTG TCG GCC CTC GGA CTC AAT GCT GTA GCG TAC TAC CGG 625 Ala Thr Lys Leu Ser Ala Leu Gly Leu Asn Ala Val Ala Tyr Tyr Arg 195 200 205 GGT CTT GAT GTG TCC G 641 Gly Leu Asp Val Ser 210 配列番号：１３ 配列の長さ：432 塩基対 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 genomic RNA 配列 CA GGC GAG AGG CCG ACA GGG ATG TTT GAC AGC GTA GTG CTC TGT GAG 47 Gly Glu Arg Pro Thr Gly Met Phe Asp Ser Val Val Leu Cys Glu 1 5 10 15 TGC TAT GAT GCC GGG GCC GCC TGG TAC GAG CTT ACG CCT GCT GAG ACT 95 Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr 20 25 30 ACG GTG AGA CTC CGG GCT TAT TTC AAC ACG CCC GGT TTG CCT GTA TGT 143 Thr Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly Leu Pro Val Cys 35 40 45 CAA GAC CAC CTA GAG TTC TGG GAA GCG GTC TTC ACA GGT CTC ACA CAC 191 Gln Asp His Leu Glu Phe Trp Glu Ala Val Phe Thr Gly Leu Thr His 50 55 60 ATT GAT GCC CAC TTC CTC TCC CAG ACG AAG CAA GGA GGA GAC AAC TTT 239 Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Gly Gly Asp Asn Phe 65 70 75 GCG TAT CTA ACG GCC TAC CAG GCC ACA GTA TGC GCC AGG GCA AAG GCC 287 Ala Tyr Leu Thr Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala 80 85 90 95 CCC CCT CCT TCG TGG GAC GTG ATG TGG AAG TGT CTA ATC AGG CTC AAA 335 Pro Pro Pro Ser Trp Asp Val Met Trp Lys Cys Leu Ile Arg Leu Lys 100 105 110 CCT ACA TTG ACT GGT CCT ACC CCC CTC CTG TAC CGC TTG GGT GCC GTG 383 Pro Thr Leu Thr Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val 115 120 125 ACT AAC GAG GTT ACC CTG ACG CAC CCC GTG ACG AAA TAT ATC GCC ACG T 432 Thr Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Ala Thr 130 135 140 SEQ ID NO: 1 Sequence length: 763 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Sequence CG CAG TCA TTC CAA GTG GCC CAT CTA CAC GCT CCC ACT GGC AGC GGC 47 Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly 1 5 10 15 AAG AGT ACT AAA GTG CCG GCT GCA TAT GCC AGC CAA GGG TAC AAG GTG 95 Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ser Gln Gly Tyr Lys Val 20 25 30 CTC GTC CTC AAC CCG TCC GTT GCC GCC ACC TTA GGT TTT GGA GCG TAT 143 Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr 35 40 45 ATG TCT AAG GCA CAT GGC ACC GAC CCC AAC ATC AGA ACT GGG GTA AGG 191 Met Ser Lys Ala His Gly Thr Asp Pro Asn Ile Arg Thr Gly Val Arg 50 55 60 ACT ATC ACC ACA GGC GCC CCC ATC ACG TAC TCC ACC TAC GGC AAG TTC 239 Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe 65 70 75 CTT GCC GAC GGT GGT TGT TCT GGG GGC GCT TAT GAC ATC ATA ATG TGT 287 Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Met Cys 8 0 85 90 95 GAT GAG TGC CAC TCA ACT GAC GCG ACT TCC ATC TTG GGC ATC GGC ACG 335 Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr 100 105 110 GTC CTG GAC CAA GCG GAG ACG GCT GGA GCA CGG CTC GTC GTG CTC GCC 383 Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala 115 120 125 ACC GCT ACG CCT CCG GGA TCG GTC ACC GTG CCA CAC CCG AAT ATT GAG 431 Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu 130 135 140 GAG GTG GCC CTG TCT AAC ACT GGA GAG ATC CCC TTC TAT GGC AAA GGC 479 Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Gly 145 150 155 ATC CCC ATT GAA GTC ATC AAG GGG GGA AGG CAT CTC ATT TTC TGC CAT 527 Ile Pro Ile Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His 160 165 170 175 TCC AAG AAG AAG TGC GAC GAG CTC GCC GCG AAG TTG TCA GGC CTC GGG 575 Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly 180 185 190 ATT AAT GCT GTG GCA TAC TAC CGG GGT CTT GAT GTG TCC GTC ATA CCG 623 Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro 195 200 205 ACC AGC GGA GAC GTC GTT GTC GTG GCA ACA GAC GCT CTA ATG ACG GGC 671 Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly 210 215 220 TAT ACC GGC GAT TTT GAC TCA GTG ATC GAC TGT AAC ACA TGC GTC ACC 719 Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr 225 230 235 CAG ACA GTC GAC TTC AGC TTG GAC CCC ACC TTC ACC ATT GAG AC 763 Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu 240 245 250 SEQ ID NO: 2 Sequence length: 615 Base pair Sequence type: Nucleic acid Strand number: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Sequence C ACG CCC GGT TTG CCC GTG TGT CAA GAC CAC CTG GAG TTC TGG GAA GCG 49 Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala 1 5 10 15 GTC TTC ACA GGT CTC ACG CAC ATT GAT GCC CAC TTC CTC TCC CAG ACA 97 Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr 20 25 30 AAG CAA GGA GGA GAC AAC TTC GCG TAT CTA ACG GCC TAC CAG GCC ACA 145 Lys Gln Gly Gly Asp Asn Phe Ala Tyr Lhe eu Thr Ala Tyr Gln Ala Thr 35 40 45 GTG TGC GCT AGG GCA AAG GCC CCT CCT CCC TCG TGG GAT GTG ATG TGG 193 Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Val Met Trp 50 55 60 AAA TGT CTA GCT AGG CTG AAG CCT ACA CTA ATT GGT CCT ACC CCC CTC 241 Lys Cys Leu Ala Arg Leu Lys Pro Thr Leu Ile Gly Pro Thr Pro Leu 65 70 75 80 CTG TAC CGC TTG GGT GCC GTG ACC AAC GAG GTT ACC CTG ACG CAC CCC 289 Leu Tyr Arg Leu Gly Ala Val Thr Asn Glu Val Thr Leu Thr His Pro 85 90 95 GTG ACG AAA TAC ATC GCC ACG TGC ATG CAA GCT GAC CTC GAG ATC ATG 337 Val Thr Lys Tyr Ile Ala Thr Cys Met Gln Ala Asp Leu Glu Ile Met 100 105 110 ACG AGC ACA TGG GTC CTA GCA GGG GGG GTG CTA GCC GCC GTG GCA GCT 385 Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu Ala Ala Val Ala Ala 115 120 125 TAC TGC CTG GCA ACC GGC TGT GTT TCC ATC ATC GGC CGC CTA CAC CTG 433 Tyr Cys Leu Ala Thr Gly Cys Val Ser Ile Ile Gly Arg Leu His Leu 130 135 140 AAT GAT CAA GTG GTT GTG ACT CCT GAC AAA GAA ATC TTA TAT GAG GCC 481 Asn Asp Gln Val Val Val Thr Pro Asp Ly s Glu Ile Leu Tyr Glu Ala 145 150 155 160 TTT GAT GAG ATG GAA GAA TGC GCC TCC AAA GCC GCC CTC ATT GAG GAA 529 Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Ala Ala Leu Ile Glu Glu 165 170 175 GGG CAG CGG ATG GCG GAG ATG CTC AAG TCT AAG ATA CAA GGC CTC CTA 577 Gly Gln Arg Met Ala Glu Met Leu Lys Ser Lys Ile Gln Gly Leu Leu 180 185 190 CAA CAG GCC ACA AGA CAG GCC CAA GAC ATA CAG CCA GC 615 Gln Gln Ala Thr Arg Gln Ala Gln Asp Ile Gln Pro 195 200 SEQ ID NO: 3 Sequence length: 771 base pairs Sequence type: Nucleic acid Strand number: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Sequence GT GAG CGA GCC TCA GGA ATG TTT GAC AGT GTA GTG CTC TGT GAG TGC 47 Glu Arg Ala Ser Gly Met Phe Asp Ser Val Val Leu Cys Glu Cys 1 5 10 15 TAT GAC GCA GGG GCT GCA TGG TAC GAG CTT ACA CCA GCG GAG ACC ACC 95 Tyr Asp Ala Gly Ala Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr 20 25 30 GTC AGG CTC AGA GCG TAT TTC AAC ACA CCT GGC TTG CCT GTG TGT CAA 143 Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly L eu Pro Val Cys Gln 35 40 45 GAC CAT CTT GAG TTC TGG GAG GCA GTT TTC ACC GGC CTC ACA CAC ATA 191 Asp His Leu Glu Phe Trp Glu Ala Val Phe Thr Gly Leu Thr His Ile 50 55 60 GAT GCC CAC TTC CTT TCC CAG ACA AAG CAA GCA GGG GAC AAT TTC GCA 239 Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Ala 65 70 75 TAC TTG ACA GCC TAC CAG GCT ACA GTG TGC GCC AGA GCC AAA GCC CCT 287 Tyr Leu Thr Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala Pro 80 85 90 95 CCC CCG TCC TGG GAC GTC ATG TGG AAG TGC CTG ACT CGG CTC AAG CCC 335 Pro Pro Ser Trp Asp Val Met Trp Lys Cys Leu Thr Arg Leu Lys Pro 100 105 110 ACG CTT GTG GCC CCT ACA CCC CTT CTG TAC CGT TTA GGC TCT GTT ACT 383 Thr Leu Val Ala Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ser Val Thr 115 120 125 AAC GAG GTC ACC CTC ACA CAT CCT GTG ACG AAA TAC ATC GCC ACT TGC 431 Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Ala Thr Cys 130 135 140 ATG CAA GCT GAC CTT GAG GTC ATG ACC AGC ACG TGG GTC CTA GCT GGG 479 Met Gln Ala Asp Leu Glu Val Met Thr Ser Thr Tr p Val Leu Ala Gly 145 150 155 GGG GTC TTG GCA GCC GTC GCC GCG TAT TGC CTG GCG ACT GGG TGT GTC 527 Gly Val Leu Ala Ala Val Ala Ala Tyr Cys Leu Ala Thr Gly Cys Val 160 165 170 175 TCC ATC ATC GGC CGC TTG CAC ATC AAT CAG CGA GCC GTC GTT GCA CCA 575 Ser Ile Ile Gly Arg Leu His Ile Asn Gln Arg Ala Val Val Ala Pro 180 185 190 GAC AAG GAG GTC CTT TAT GAG GCT TTT GAT GAG ATG GAG GAG TGT GCC 623 Asp Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala 195 200 205 TCT AAA GCG GCT CTC ATT GAA GAG GGG CAG CGG ATA GCC GAG ATG CTG 671 Ser Lys Ala Ala Leu Ile Glu Glu Gly Gln Arg Ile Ala Glu Met Leu 210 215 220 AAG TCC AAG ATC CAA GGC TTA TTG CAG CAA GCC TCT AAA CAG GCC CAG 719 Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser Lys Gln Ala Gln 225 230 235 GAC ATA CAA CCC GCT GTG CAG CCT CAT GGC CCA AGG TGG AGC AAT TCT 767 Asp Ile Gln Pro Ala Val Gln Pro His Gly Pro Arg Trp Ser Asn Ser 240 245 250 255 GGG C 771 Gly SEQ ID NO: 4 Sequence length: 630 Base pair Sequence type: Number of nucleic acid strands Double-stranded topology: Linear Sequence type: cDNA to genomic RNA sequence C TGG TAT GAA CTT ACG CCT GCT GAG ACT ACG GTG AGA CTC CGG GCC TAT 49 Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr 1 5 10 15 TTC AAC ACG CCC GGC CTG CCT GTG TGT CAA GAC CAC CTG GAA TTC TGG 97 Phe Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp 20 25 30 GAG GCG GTC TTC ACA GGT CTC ACA CAC ATC GAT GCC CAC TTC CTC TCC 145 Glu Ala Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser 35 40 45 CAG ACG AAG CAA GGA GGA GAT AAC TTT GCA TAT TTA ACA GCC TAC CAG 193 Gln Thr Lys Gln Gly Gly Asp Asn Phe Ala Tyr Leu Thr Ala Tyr Gln 50 55 60 GCC ACA GTC TGC GCT AGG GCA AAG GCT CCC CCT CCT TCG TGG GAC GTG 241 Ala Thr Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Val 65 70 75 80 ATG TGG AAG TGT TTG ATT AGG CTC AAA CCT ACA CTG ACT GGT CCT ACC 289 Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu Thr Gly Pro Thr 85 90 95 CCC CTC CTG TAC CGC TTG GGT GCC GTG ACC AAC GAG GTT ACC CTG ACT 3 37 Pro Leu Leu Tyr Arg Leu Gly Ala Val Thr Asn Glu Val Thr Leu Thr 100 105 110 CAC CCC ATG ACG AAA TAT ATC GCC ACT TGT ATG CAA GCT GAT CTT GAG 385 His Pro Met Thr Lys Tyr Ile Ala Thr Cys Met Gln Ala Asp Leu Glu 115 120 125 ATC ATG ACA AGC ACA TGG GTC TTG GCG GGG GGG GTG CTA GCC GCT GTG 433 Ile Met Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu Ala Ala Val 130 135 140 GCA GCT TAC TGC CTA GCG ACC GGC TGC ATT TCC ATC ATT GGC CGC CTT 481 Ala Ala Tyr Cys Leu Ala Thr Gly Cys Ile Ser Ile Ile Gly Arg Leu 145 150 155 160 CAC CTG AAT GAT CGG GTG GTC GTG ACC CCT GAT AAG GAA ATT TTA TAT 529 His Leu Asn Asp Arg Val Val Val Thr Pro Asp Lys Glu Ile Leu Tyr 165 170 175 GAG GCC TTT GAT GAG ATG GAA GAG TGC GCC TCC AAA GCC GCC CTC ATT 577 Glu Ala Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Ala Ala Leu Ile 180 185 190 GAG GAA GGG CAG CGG ATG GCG GAG ATG CTG AAG TCT AAA ATA CAA GGC 625 Glu Glu Gly Gln Arg Met Ala Glu Met Leu Lys Ser Lys Ile Gln Gly 195 200 205 CTC TT 630 Leu SEQ ID NO: 5 Sequence length 1426 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Sequence GGG ATC AAC CCT AAC ATC AGG ACC GGA GTA CGG ACC GTG ACC ACC GGG 48 Gly Ile Asn Pro Asn Ile Arg Thr Gly Val Arg Thr Val Thr Thr Gly 1 5 10 15 GAC TCC ATC ACC TAC TCC ACT TAT GGC AAG TTT ATC GCA GAT GGA GGT 96 Asp Ser Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Ile Ala Asp Gly Gly 20 25 30 TGC GCA CAT GGT GCC TAT GAC GTC ATC ATA TGC GAC GAA TGC CAT TCA 144 Cys Ala His Gly Ala Tyr Asp Val Ile Ile Cys Asp Glu Cys His Ser 35 40 45 GTG GAC GCT ACT ACC ATC CTT GGC ATT GGA ACA GTC CTT GAC CAG GCT 192 Val Asp Ala Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala 50 55 60 GAG ACC GCA GGT GCC AGG CTA GTG GTT TTA GCC ACA GCC ACG CCA CCC 240 Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro 65 70 75 80 GGT ACG GTA ACA ACT CCC CAC GCT AAC ATA GAG GAG GTG GCC CTT GGT 288 Gly Thr Val Thr Thr Pro His Ala Asn Ile Glu Glu Val Ala Leu Gly 85 90 95 CAC GAA GGC GAG ATT CCT TTT TAT GGC AAG GCT ATT CCC CTA GCT TTC 336 His Glu Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Ala Phe 100 105 110 ATC AAG GGG GGC AGA CAC CTA ATT TTT TGC CAT TCA AAG AAG AAG TGC 384 Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys 115 120 125 GAC GAG CTC GCA GCA GCC CTT CGG GGC ATG GGT ATC AAT GCC GTT GCC 432 Asp Glu Leu Ala Ala Ala Leu Arg Gly Met Gly Ile Asn Ala Val Ala 130 135 140 TAC TAC AGG GGT CTC GAC GTC TCC GTT ATA CCA ACT CAA GGA GAC GTG 480 Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Gln Gly Asp Val 145 150 155 160 GTG GTT GTC GCC ACC GAT GCC CTA ATG ACT GGA TAC ACC GGT GAC TTT 528 Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe 165 170 175 GAC TCT GTC ATC GAC TGC AAC GTT GCA GTC ACT CAG ATT GTT GAC TTT 576 Asp Ser Val Ile Asp Cys Asn Val Ala Val Thr Gln Ile Val Asp Phe 180 185 190 AGC CTA GAC CCA ACT TTT ACC ATC ACC ACT CAA ACC GTC CCT CAG GAG 624 Ser Leu Asp Pro Thr Phe Thr Ile Thr Thr Gln Thr Val Pro Gln Glu 195 200 205 GCT GTCTCC CGT AGT CAA CGT AGA GGG AGA ACT GGG AGG GGG CGA CTG 672 Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Leu 210 215 220 GGC ACT TAC AGG TAT GTC TCG TCA GGC GAG AGG CCG TCT GGG ATG TTC 720 Gly Thr Tyr Arg Tyr Val Ser Ser Gly Glu Arg Pro Ser Gly Met Phe 225 230 235 240 GAC AGC GTA GTA CTC TGC GAG TGC TAT GAT GCC GGG GCA GCC TGG TAC 768 Asp Ser Val Val Leu Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr 245 250 255 GAG CTT ACA CCT GCT GAG ACC ACA GTG AGA CTC CGG GCT TAT TTC AAC 816 Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Phe Asn 260 265 270 ACG CCC GGT TTG CCC GTG TGT CAA GAC CAC CTG GAG TTC TGG GAA GCG 864 Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala 275 280 285 GTC TTC ACA GGT CTC ACG CAC ATT GAT GCC CAC TTC CTC TCC CAG ACA 912 Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr 290 295 300 AAG CAA GGA GGA GAC AAC TTC GCG TAT CTA ACG GCC TAC CAG GCC ACA 960 Lys Gln Gly Gly Asp Asn Phe Ala Tyr Leu Thr Ala Tyr Gln Ala Thr 305 310 315 320 GTG TGC GCT AGG GCA AAG GCC CCT CCT CCC TCG TGG GAT GTG ATG TGG 1008 Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Val Met Trp 325 330 335 AAA TGT CTA GCT AGG CTG AAG CCT ACA CTA ATT GGT CCT ACC CCC CTC 1056 Lys Cys Leu Ala Arg Leu Lys Pro Thr Leu Ile Gly Pro Thr Pro Leu 340 345 350 CTG TAC CGC TTG GGT GCC GTG ACC AAC GAG GTT ACC CTG ACG CAC CCC 1104 Leu Tyr Arg Leu Gly Ala Val Thr Asn Glu Val Thr Leu Thr His Pro 355 360 365 GTG ACG AAA TAC ATC GCC ACG TGC ATG CAA GTG AAC CTC GAG ATC ATG 1152 Val Thr Lys Tyr Ile Ala Thr Cys Met Gln Val Asn Leu Glu Ile Met 370 375 380 ACG AGC ACA TGG GTC CTA GCA GGG GGG GTG CTA GCC GCC GTG GCA GCT 1200 Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu Ala Ala Val Ala Ala 385 390 395 395 400 TAC TGC CTG GCA ACC GGC TGT GTT TCC ATC ATC GGC CGC CTA CAC CTG 1248 Tyr Cys Leu Ala Thr Gly Cys Val Ser Ile Ile Gly Arg Leu His Leu 405 410 415 AAT GAT CAA GTG GTT GTG ACT CCT GAC AAA GAA ATC TTA TAT GAG GCC 1296 Asn Asp Gln Val Val Val Thr Pro Asp Lys Glu Ile Leu Tyr Glu Ala 420 425 430 TTT GAT GAG ATG GAA GAA TGC GCC TCC AAA GCC GCC CTC ATT GAG GAA 1344 Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Ala Ala Leu Ile Glu Glu 435 440 445 GGG CAG CGG ATG GCG GAG ATG CTC AAG TCT AAG ATA CAA GGC CTC CTA 1392 Gly Gln Arg Met Ala Glu Met Leu Lys Ser Lys Ile Gln Gly Leu Leu 450 455 460 CAA CAG GCC ACA AGA CAG GCC CAA GAC ATA CAG C 1426 Gln Gln Ala Thr Arg Gln Ala Gln Asp Ile Gln 465 470 475 SEQ ID NO: 6 Sequence length: 855 Base pair Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Sequence CG CAG ACA TTC CAA GTG GCC CAT CTG CAC GCT CCC ACT GGT AGC GGC 47 Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly 1 5 10 15 AAG AGC ACT AAG GTG CCG GCT GCA TAT GCG GCC CAA GGG TAC AAG GTA 95 Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val 20 25 30 CTC GTC CTG AAC CCG TCC GTT GCC GCC ACT TTA GCC TTT GGG GCG TAC 143 Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Ala Phe Gly Ala Ty r 35 40 45 ATG TCT AAG GCA CAT GGT GTC GAC CCT AAC ATC AGA ACT GGG GTG AGG 191 Met Ser Lys Ala His Gly Val Asp Pro Asn Ile Arg Thr Gly Val Arg 50 55 60 ACC ATC ACC ACG GGC GCT CCC ATC ACG TAC TCC ACC TAT GGT AAG TTC 239 Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe 65 70 75 CTT GCC GAC GGT GGT TGC TCT GGG GGC GCC TAT GAC ATC ATA ATA TGT 287 Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys 80 85 90 95 GAT GAG TGC CAC TCA ACT GAC TCG ACA TCC ATC TTG GGC ATC GGC ACA 335 Asp Glu Cys His Ser Thr Asp Ser Thr Ser Ile Leu Gly Ile Gly Thr 100 105 110 GTC CTG GAC CAA GCG GAG ACG GCT GGA GCG CGG CTC GTC GTG CTC GCT 383 Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala 115 120 125 ACC GCT ACG CCT CCG GGA TCG GTC ACC GTG CCA CAT CCC AAT ATC GAG 431 Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu 130 135 140 GAG GTG GCC CTG TCC ACC ACT GGA GAG ATT CCC TTC TAC GGC AAA GCT 479 Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala 145 150 155 ATC CCC ATC GAG ACA ATC AAG GGG GGG AGG CAT CTC ATC TTC TGC CGT 527 Ile Pro Ile Glu Thr Ile Lys Gly Gly Arg His Leu Ile Phe Cys Arg 160 165 170 175 TCC AAG AAG AAG TGT GAC GAG CTC GCT GGA AAG CTG TCA GCC CTC GGA 575 Ser Lys Lys Lys Cys Asp Glu Leu Ala Gly Lys Leu Ser Ala Leu Gly 180 185 190 ATC AAC GCT GTA GCG TAC TAC CGG GGT CTT GAT GTA TCC GTC ATA CCG 623 Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro 195 200 205 ACC AGC GGA GAC GTC GTT GTC GTG GCA ACA GAC GCT CTA ATG ACG GGC 671 Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly 210 215 220 TAC ACC GGT GAC TTT GAT TCA GTG ATC GAC TGC AAT ACA TGT GTC ACC 719 Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr 225 230 235 CAG ACA GTC GAC TTC AGC TTG GAC CCT ACC TTC ACC ATT GAG ACG ACG 767 Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr 240 245 250 255 ACC GTG CCT CAA GAC GCG GTG TCA CGC TCG CAG CGG CGA GGC AGA ACT 815 Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Ar g Arg Gly Arg Thr 260 265 270 GGT AGG GGT AGA GGG GGC ATA TAC AGG TTT GTG ACT CCA G 855 Gly Arg Gly Arg Gly Gly Ile Tyr Arg Phe Val Thr Pro 275 280 SEQ ID NO: 7 Sequence length: 315 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Sequence GAC GAG CTC GCC GCA AAG CTG TCA GGC CTC GGA GTC AAT GCT GTG GCA 48 Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Val Asn Ala Val Ala 1 5 10 15 TAC TAC CGG GGT CTC GAT GTG TCT GTC ATA CCG ACG AGC GGG GAC GTC 96 Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val 20 25 30 GTT GTT GTG GCA ACA GAC GCT CTA ATG ACG GGC TAT ACC GGC GAC TTT 144 Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe 35 40 45 GAC TCG GTG ATC GAC TGC AAT ACA TGT GTC ACC CAA ACA GTC GAT TTC 192 Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe 50 55 60 AGC TTG GAC CCT ACT TTC ACC ATT GAG ACG ACG ACC GTG CCC CAA GAC 240 Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Thr Val Pro Gln Asp 65 70 75 80 GCG GTG TCG CGC TCG CAG CGG CGA GGC AGG ACT GGT AGG GGC AGG GTG 288 Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Val 85 90 95 GGC ATA TAC AGG TTT GTG ACT CCC GAG 315 Gly Ile Tyr Arg Phe Val Thr Pro Glu 100 105 SEQ ID NO: 8 Sequence length: 911 base pairs Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA sequence GT GAT GAG CTC GCC GCA AAG CTC TCA AGC CTC GGA CTC AAC GCT GTA 47 Asp Glu Leu Ala Ala Lys Leu Ser Ser Leu Gly Leu Asn Ala Val 1 5 10 15 GCA TAT TAC CGG GGT CTT GAT GTG TCC GTC ATA CCG ACT AGT GGA GAC 95 Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp 20 25 30 GTC GTT GTC GTG GCA ACA GAC GCT CTA ATG ACG GGC TAT ACC GGC GAC 143 Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp 35 40 45 TTT GAC TCA GTG ATC GAC TGT AAC ACA TGT GTC ACC CAG ACA GTT GAT 191 Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp 50 55 60 TTC AGC TTG GAT CCA ACC TTC ACC ATT GAG ACG ACG ACC GTG CCT CAA 239 Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln 65 70 75 GAC GCG GTG TCG CGC TCG CAG CGG CGA GGT AGG ACT GGC AGG GGC AGG 287 Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg 80 85 90 95 GGC GGC ATC TAT AGG TTT GTG ACT CCA GGA GAA CGG CCC TCG GGC ATG 335 Gly Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met 100 105 110 TTC GAT TCC TCG GTC CTG TGT GAG TGT TAT GAC GCG GGC TGT GCT TGG 383 Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp 115 120 125 TAT GAG CTC ACG CCC GCC GAG ACC ACG GTT AGG TTG CGG GCT TAC CTA 431 Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Leu 130 135 140 AAT ACA CCA GGG TTG CCC GTC TGC CAG GAC CAT CTG GAG TTC TGG GAG 479 Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu 145 150 155 GGC GTC TTC ACA GGC CTC ACC CAC ATA GAT GCC CAT TTC TTG TCT CAG 527 Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln 160 165 170 175 ACT AAG CAG GC A GGA CAC AAC TTT CCC TAC CTG GTG GCA TAC CAA GCT 575 Thr Lys Gln Ala Gly His Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala 180 185 190 ACA GTG TGC GCC AGG GCT CAG GCT CCA CCT CCA TCG TGG GAC CAA ATG 623 Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met 195 200 205 TGG AAG TGT CTC ATA CGG CTG AAA CCT ACG CTG CAC GGG CCA ACA CCC 671 Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro 210 215 220 CTG CTG TAT AGG CTA GGA GCC GTG GAA AAT GAG GTC ACC CTC ACA CAC 719 Leu Leu Tyr Arg Leu Gly Ala Val Glu Asn Glu Val Thr Leu Thr His 225 230 235 CCC ATA ACC AAA TTC ATC ATG GCA TGC ATG TCG GCT GAT CTG GAG GTC 767 Pro Ile Thr Lys Phe Ile Met Ala Cys Met Ser Ala Asp Leu Glu Val 240 245 250 255 GTC ACC AGC ACC TGG GTG CTG GTG GGC GGA GTC CTT GCA GCT CTG GCC 815 Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala 260 265 270 GCA TAT CGC CTG ACA ACA GGC AGC GTG GTC ATC GTG GGT AGG ATC ATC 863 Ala Tyr Arg Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile 275 280 285 TT G TCT GGG AGG CCG GCT GTC ATT CCC GAC AGG GAA GTC CTT TAC CGG 911 Leu Ser Gly Arg Pro Ala Val Ile Pro Asp Arg Glu Val Leu Tyr Arg 290 295 300 SEQ ID NO: 9 Sequence length: 489 Base pair sequence Type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Sequence CG ACA ACC GTG CCC CAA GAC GCG GTG TCG CGC TCA CAA CGG CGG GGT 47 Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly 1 5 10 15 AGG ACA GGT AGG GGC AGG AGA GGC ATC TAC AGA TTT GTG ACT CCG GGA 95 Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly 20 25 30 GAA CGG CCC TCG GGC ATG TTC GAT TCT TCG GTC CTG TGT GAG TGC TAT 143 Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr 35 40 45 GAC GCG GGC TGC GCT TGG ATC GAG CTC ACG CCC GCC GAG ACC TCA GTT 191 Asp Ala Gly Cys Ala Trp Ile Glu Leu Thr Pro Ala Glu Thr Ser Val 50 55 60 AGG TTG CGG GCT TAC CTA AAT ACA CCA GGG TTG CCC GTC TGC CAG GAC 239 Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp 65 70 75 CAC CTG GAA TTC TGG GAG AGC GTC TTC ACA GGC CTC ACC CAT ATA GAT 287 His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp 80 85 90 95 GCC CAC TTC TTG TCC CAG ACC AAG CAG GCA GGA GAC AAC TTC CCC TAC 335 Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr 100 105 110 CTG GTA GCA TAC CAA GCT ACA GTG TGC GCC AGG GCC CAG GCT CCA CCA 383 Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro 115 120 125 CCA TCG TGG GAT CAA ATG TGG AAG TGT CTC ATA CGG CTG AAA CCT ACG 431 Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr 130 135 140 CTA CAC GGG CCA ACA CCC CTG TTG TAT AGG CTG GGA GCC GTC CAA AAT 479 Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn 145 150 155 GAG GTC ACC C 489 Glu Val Thr 160 SEQ ID NO: 10 Sequence length Length: 1076 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Sequence GT GGT CTC CTG GGT GCC ATC GTG GTC AGC CTA ACG GGC CGC GAC AAG 47 Gly Leu Leu Gly Ala Ile Val Val Ser Leu Thr Gly Arg Asp Lys 1 5 10 15 AAC CAG GTC GAG GGG GAG GTT CAG GTG GTC TCC ACC GCA ACG CAA TCT 95 Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser 20 25 30 TTC CTG GCG ACC TGC GTC AAT GGC GTG TGT TGG ACC GTC TAC CAT GGC 143 Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His Gly 35 40 45 GCC GGC TCG AAA ACC CTG GCC GGC CCG AAG GGT CCA GTC ACC CAA ATG 191 Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Val Thr Gln Met 50 55 60 TAC ACT AAT GTG GAC CAG GAC CTC GTC GGC TGG CCG GCG CCC TCC GGG 239 Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Ser Gly 65 70 75 GCG CGG TCC TTG ACA CCA TGC ACC TGC GGC AGC TCG GAC CTT TAC TTG 287 Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu 80 85 90 95 GTC ACG AGG CAT GCT GAT GTC ATT CCG GTG CGC CGG CGG GGC GAT AGC 335 Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser 100 105 110 AGG GGG AGC CTG CTT TCC CCC AGG CCC CTC TCC TAC TTG AAG GGC TCC 383 Arg Gly Se r Leu Leu Ser Pro Arg Pro Leu Ser Tyr Leu Lys Gly Ser 115 120 125 TCA GGT GGT CCA CTG CTT TGC CCC TCG GGG CAC ATT GTG GGC ATC TTC 431 Ser Gly Gly Pro Leu Leu Cys Pro Ser Gly His Ile Val Gly Ile Phe 130 135 140 CGG GCT GCC GTG TGC ACC CGG GGG GTT GCG AAG GCG GTG GAC TTT GTA 479 Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val 145 150 155 CCT GTC GAG TCT ATG GAA ACT ACT ATG CGG TCT CCG GTC TTC ACG GAT 527 Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp 160 165 170 175 AAT TCA TCC CCC CCG GCC GTA CCG CAG ACA TTC CAA GTG GCC CAT CTG 575 Asn Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu 180 185 190 CAT GCC CCC ACT GGC AGC GGC AAG AGC ACT AAG GTG CCG GCT GCA TAC 623 His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr 195 200 205 GCA GCC CAG GGA TAC AAG GTA CTC GTC CTG AAC CCG TCC GTT GCC GCC 671 Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala 210 215 220 ACC TTA GGT TTT GGA GCA TAT ATG TCC AAG GCA CAT GGT GTC GAC CCT 71 9 Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro 225 230 235 AAC ATC AGG ACT GGG GTA AGG ACC ATC ACT ACG GGC GCC CCC ATT ACA 767 Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr 240 245 250 255 TAC TCC ACC TAT GGC AAG TTT CTT GCC GAC GGT GGT TGC TCC GGG GGC 815 Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly 260 265 270 GCC TAT GAC ATC ATA ATA TGT GAT GAG TGC CAC TCA ACT GAC TCG ACT 863 Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr 275 280 285 TCC ATT TTG GGC ATT GGC ACG GTC CTG GAC CAA GCG GAG ACG GCT GGA 911 Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly 290 295 300 GCG CGG CTC GTC GTG CTC GCC ACC GCT ACG CCT CCA GGA TCG GTC ACT 959 Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr 305 310 315 GTG CCT CAT CCC AAC ATC GAG GAG GTG GCC TTG TCC AGC ACT GGA GAG 1007 Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Ser Thr Gly Glu 320 325 330 335 ATT CCC TTC TAT GGC AAA GCC ATC CCC ATT GAG ACC A TC AAG GGG GGA 1055 Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Thr Ile Lys Gly Gly 340 345 350 AGG CAT CTC ATT TTC TGC CAC 1076 Arg His Leu Ile Phe Cys His 355 SEQ ID NO: 11 Sequence length: 284 Base pair Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA sequence GTC GAC CCC AAT ATT AGA ACT GGG GTA AGG ACC ATC ACC ACG GGC GCT 48 Val Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala 1 5 10 15 CCC ATT ACG TAT TCT ACC TAT GGC AAA TTC CTT GCC GAC GGT GGT TGC 96 Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys 20 25 30 TCT GGG GGC GCC TAT GAC ATC ATA ATC TGT GAT GAG TGC CAC TCA ACT 144 Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr 35 40 45 GAC TCG ACT TCC ATC TTG GGT ATC GGC ACA GCC CTG GAC CAA GCG GAG 192 Asp Ser Thr Ser Ile Leu Gly Ile Gly Thr Ala Leu Asp Gln Ala Glu 50 55 60 ACG GCT GGA GCA CGG CTT GTC GTG CTC GCC ACC GCT ACG CCT CCA GGG 240 Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly 65 70 75 80 TCG GTC ACC GTG CCG CAT CCC AAC ATC GAG GAG GTA GCC TTG CC 284 Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu 85 90 SEQ ID NO: 12 Sequence length Length: 641 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA sequence G GAC AAC TCA TCT CCC CCG GCG GTA CCG CAG ACA TTC CAG GTG GCC CAT 49 Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His 1 5 10 15 CTA CAC GCT CCC ACT GGC AGC GGC AAG AGC ACT AAG GTG CCG GCT GCA 97 Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala 20 25 30 TAT GCA GCC CAA GGG TAC AAA GTA CTC GTC CTG AAC CCG TCC GTT GCC 145 Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala 35 40 45 GCC ACC TTA AGT TTC GGG GCG TAT ATG TCC AAG GCA CAT GGT GTT GAC 193 Ala Thr Leu Ser Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp 50 55 60 CCT AAT ATC AGA ACT GGG ACA AGG ACC ATC ACC ACG GGC GCT CCC ATC 241 Pro Asn Ile Arg Thr Gly Thr A rg Thr Ile Thr Thr Gly Ala Pro Ile 65 70 75 80 ACG TAC TCC ACC TAT GGC AAG TTC CTT GCA GAC GGT GGT TGC TCC GGA 289 Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly 85 90 95 GGC GCC TAT GAC ATC ATA ATA TGC GAT GAG TGC CAC TCA ACA GAC TCG 337 Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser 100 105 110 ACT TCC ATC TTA GGC ATT GGT ACG GTC CTG GAC CAA GCG GAG ACG GCT 385 Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala 115 120 125 GGA GCG CGA CTC GTC GTG CTC GCC ACC GCT ACG CCT CCA GGA TCG GTC 433 Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val 130 135 140 ACT GTG CCA CAT CCC AAC ATC GAG GAG GTG GCC CTG TCC AAC ACT GGA 481 Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly 145 150 155 160 GAG ATT CCC TTC TAT GGC AAA GCC ATC CCC ATT GAG GCC ATC AAG GGG 529 Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Ala Ile Lys Gly 165 170 175 GGG AGG CAT CTC ATT TTC TGC CAT TCT AAG AAG AAG TGT GAT GAG CTC 577 Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu 180 185 190 GCC ACG AAG CTG TCG GCC CTC GGA CTC AAT GCT GTA GCG TAC TAC CGG 625 Ala Thr Lys Leu Ser Ala Leu Gly Leu Asn Ala Val Ala Tyr Tyr Arg 195 200 205 GGT CTT GAT GTG TCC G 641 Gly Leu Asp Val Ser 210 SEQ ID NO: 13 Sequence length: 432 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Sequence CA GGC GAG AGG CCG ACA GGG ATG TTT GAC AGC GTA GTG CTC TGT GAG 47 Gly Glu Arg Pro Thr Gly Met Phe Asp Ser Val Val Leu Cys Glu 1 5 10 15 TGC TAT GAT GCC GGG GCC GCC TGG TAC GAG CTT ACG CCT GCT GAG ACT 95 Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr 20 25 30 ACG GTG AGA CTC CGG GCT TAT TTC AAC ACG CCC GGT TTG CCT GTA TGT 143 Thr Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly Leu Pro Val Cys 35 40 45 CAA GAC CAC CTA GAG TTC TGG GAA GCG GTC TTC ACA GGT CTC ACA CAC 191 Gln Asp His Leu Glu Phe Trp Glu Ala Val Phe Thr Gly Leu Thr His 50 55 60 ATT GAT GCC CAC TTC CTC TCC CAG ACG AAG CAA GGA GGA GAC AAC TTT 239 Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Gly Gly Asp Asn Phe 65 70 75 GCG TAT CTA ACG GCC TAC CAG GCC ACA GTA TGC GCC AGG GCA AAG GCC 287 Ala Tyr Leu Thr Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Lys Ala 80 85 90 95 CCC CCT CCT TCG TGG GAC GTG ATG TGG AAG TGT CTA ATC AGG CTC AAA 335 Pro Pro Pro Ser Trp Asp Val Met Trp Lys Cys Leu Ile Arg Leu Lys 100 105 110 CCT ACA TTG ACT GGT CCT ACC CCC CTC CTG TAC CGC TTG GGT GCC GTG 383 Pro Thr Leu Thr Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val 115 120 125 ACT AAC GAG GTT ACC CTG ACG CAC CCC GTG ACG AAA TAT ATC GCC ACG T 432 Thr Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Ala Thr 130 135 140
【図１】第１図は、発現プラスミド Trp・TrpE・C11-7
の構築方法を示す。FIG. 1 shows expression plasmids Trp / TrpE / C11-7.
The construction method of is shown.
【図２】第２図は、発現産物と正常人血清または非Ａ非
Ｂ型肝炎患者血清との免疫反応性をウエスタンブロット
法により調べた結果を示す写真である。 Ａ…精製抗原， Ｂ…発現菌体抽出物， Ｃ…非発現菌
体抽出物。FIG. 2 is a photograph showing the results of examining the immunoreactivity of the expression product with normal human serum or non-A non-B hepatitis patient serum by Western blotting. A ... Purified antigen, B ... Extract of expressing cells, C ... Extract of non-expressing cells.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｎ 15/10 15/11 15/71 Ｃ１２Ｐ 21/02 Ｃ 8214−4Ｂ 21/08 8214−4Ｂ Ｃ１２Ｑ 1/68 ＺＮＡ Ａ 8114−4Ｂ Ｇ０１Ｎ 33/53 Ｄ 8310−2Ｊ 33/569 Ｌ 9015−2Ｊ 33/576 Ｚ 9015−2Ｊ //(Ｃ１２Ｎ 1/21 Ｃ１２Ｒ 1:19） (Ｃ１２Ｐ 21/02 Ｃ１２Ｒ 1:19） (Ｃ１２Ｐ 21/08 Ｃ１２Ｒ 1:91） (72)発明者 小原 道法 埼玉県入間郡大井町西鶴ケ岡一丁目３番１ 号 東燃株式会社総合研究所内─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Office reference number FI Technical display location C12N 15/10 15/11 15/71 C12P 21/02 C 8214-4B 21/08 8214-4B C12Q 1/68 ZNA A 8114-4B G01N 33/53 D 8310-2J 33/569 L 9015-2J 33/576 Z 9015-2J // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1: 19) (C12P 21/08 C12R 1:91) (72) Inventor Michiho Ohara 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Tonen Research Institute
Claims (27)
れた非Ａ非Ｂ型肝炎ウイルスＲＮＡから遺伝子工学的に
誘導して得られたＤＮＡ断片であって、該ウイルス非構
造タンパク質由来の非Ａ非Ｂ型肝炎特異抗原ポリペプチ
ドをコードする塩基配列を含むことを特徴とするＤＮＡ
断片。1. A DNA fragment obtained by genetically inducing non-A non-B hepatitis virus RNA directly obtained from plasma of a non-A non-B hepatitis patient, which is derived from the virus non-structural protein. DNA comprising a base sequence encoding a non-A non-B hepatitis specific antigen polypeptide
fragment.
て示される読み取り枠に従ってコードされるアミノ酸配
列の全部又は一部で表わされることを特徴とする請求項
１記載のＤＮＡ断片。2. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of an amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 1.
て示される読み取り枠に従ってコードされるアミノ酸配
列の全部又は一部で表わされることを特徴とする請求項
１記載のＤＮＡ断片。3. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 2.
て示される読み取り枠に従ってコードされるアミノ酸配
列の全部又は一部で表わされることを特徴とする請求項
１記載のＤＮＡ断片。4. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 3.
て示される読み取り枠に従ってコードされるアミノ酸配
列の全部又は一部で表わされることを特徴とする請求項
１記載のＤＮＡ断片。5. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 4.
て示される読み取り枠に従ってコードされるアミノ酸配
列の全部又は一部で表わされることを特徴とする請求項
１記載のＤＮＡ断片。6. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of an amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 5.
て示される読み取り枠に従ってコードされるアミノ酸配
列の全部又は一部で表わされることを特徴とする請求項
１記載のＤＮＡ断片。7. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 6.
て示される読み取り枠に従ってコードされるアミノ酸配
列の全部又は一部で表わされることを特徴とする請求項
１記載のＤＮＡ断片。8. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 7.
て示される読み取り枠に従ってコードされるアミノ酸配
列の全部又は一部で表わされることを特徴とする請求項
１記載のＤＮＡ断片。9. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of an amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 8.
って示される読み取り枠に従ってコードされるアミノ酸
配列の全部又は一部で表わされることを特徴とする請求
項１記載のＤＮＡ断片。10. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 9.
よって示される読み取り枠に従ってコードされるアミノ
酸配列の全部または一部で表わされることを特徴とする
請求項１記載のＤＮＡ断片。11. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 10.
よって示される読み取り枠に従ってコードされるアミノ
酸配列の全部または一部で表わされることを特徴とする
請求項１記載のＤＮＡ断片。12. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 11.
よって示される読み取り枠に従ってコードされるアミノ
酸配列の全部または一部で表わされることを特徴とする
請求項１記載のＤＮＡ断片。13. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 12.
よって示される読み取り枠に従ってコードされるアミノ
酸配列の全部または一部で表わされることを特徴とする
請求項１記載のＤＮＡ断片。14. The DNA fragment according to claim 1, wherein the polypeptide is represented by all or part of the amino acid sequence encoded according to the open reading frame represented by SEQ ID NO: 13.
のＤＮＡ断片を、プロモーターの下流に存在するベクタ
ー内のクローニング部位に導入して得られる発現ベクタ
ー。15. An expression vector obtained by introducing the DNA fragment according to any one of claims 1 to 14 into a cloning site in a vector existing downstream of a promoter.
５記載の発現ベクター。16. The vector according to claim 1, which is a plasmid.
The expression vector according to 5.
発現ベクターで形質転換して得られる形質転換体。17. A transformant obtained by transforming a host with the expression vector according to claim 15 or 16.
形質転換体。18. The transformant according to claim 17, wherein the host is Escherichia coli.
のＤＮＡ断片を構成する塩基配列によってコードされる
アミノ酸配列の全部または一部を含む組換えポリペプチ
ドの製造方法であって、 前記ＤＮＡ断片を適当な宿主細胞内で発現させ得る複製
可能な発現ベクターを構築する工程、 前記発現ベクターを宿主細胞内に移入して形質転換体を
得る工程、 前記ＤＮＡ断片を発現させ得る条件下で前記形質転換体
を培養して前記組換えポリペプチドを産生させる工程、 前記組換えポリペプチドを回収する工程、を含む方法。19. A method for producing a recombinant polypeptide containing all or part of an amino acid sequence encoded by the nucleotide sequence constituting the DNA fragment according to any one of claims 1 to 14, Constructing a replicable expression vector capable of expressing a DNA fragment in a suitable host cell, transferring the expression vector into a host cell to obtain a transformant, under conditions capable of expressing the DNA fragment A method comprising culturing the transformant to produce the recombinant polypeptide, and recovering the recombinant polypeptide.
のＤＮＡ断片を構成する塩基配列によってコードされる
アミノ酸配列の全部又は一部を含む組換え非Ａ非Ｂ型肝
炎特異抗原ポリペプチド。20. A recombinant non-A non-B hepatitis-specific antigen polypeptide comprising all or part of the amino acid sequence encoded by the nucleotide sequence constituting the DNA fragment according to any one of claims 1 to 14. ..
のＤＮＡ断片を構成する部分塩基配列に基づいて合成さ
れたセンスおよびアンチセンスを含む塩基配列を用い
て、非Ａ非Ｂ型肝炎ウイルス遺伝子を増幅する遺伝子増
幅法。21. A non-A non-B hepatitis is obtained using a base sequence containing sense and antisense synthesized based on the partial base sequence constituting the DNA fragment according to any one of claims 2 to 14. A gene amplification method for amplifying viral genes.
配列に基づいて合成された下記の塩基配列： ５′−ＧＧＡＴＡＣＡＣＣＧＧＴＧＡＣＴＴＴＧＡ−３′ を有する、ＰＣＲプライマー用一本鎖ＤＮＡ配列。22. A single-stranded DNA sequence for PCR primer, which has the following base sequence synthesized based on the partial base sequence of the DNA fragment according to claim 6: 5'-GGATACACCGGGTGACTTTGA-3 '.
基配列に基づいてアンチセンスになるように合成された
下記の塩基配列： ５′−ＴＧＣＡＴＧＣＡＣＧＴＧＧＣＧＡＴＧＴＡ−３′ を有する、ＰＣＲプライマー用一本鎖ＤＮＡ配列。23. A single strand for PCR primer, which has the following base sequence synthesized so as to be antisense based on the partial base sequence in the DNA fragment according to claim 6: 5′-TGCATGCACCGTGGCGATGTA-3 ′ DNA sequence.
か一項に記載のＤＮＡ断片中の部分塩基配列に基づいて
合成された下記の塩基配列： ５′−ＧＡＴＧＣＣＣＡＣＴＴＣＣＴＣＴＣＣＣＡ−３′ を有する、ＰＣＲプライマー用一本鎖ＤＮＡ配列。24. Having the following base sequence synthesized based on the partial base sequence in the DNA fragment according to any one of claims 3, 5, 6 and 14, 5'-GATGCCCACTTCCTCTCCCA-3 ', Single-stranded DNA sequence for PCR primers.
に記載のＤＮＡ断片中の部分塩基配列に基づいてアンチ
センスになるように合成された下記の塩基配列： ５′−ＧＴＣＡＧＧＧＴＡＡＣＣＴＣＧＴＴＧＧＴ−３′ を有する、ＰＣＲプライマー用一本鎖ＤＮＡ配列。25. The following base sequence synthesized so as to be antisense based on the partial base sequence in the DNA fragment according to any one of claims 3, 5 and 6: 5'-GTCAGGGTAACCTCGTGTG-3 A single-stranded DNA sequence for a PCR primer having a '.
子を検出するための方法であって、 検体からＲＮＡを取り出す段階、 得られたＲＮＡに逆転写酵素を作用させてｃＤＮＡを合
成する段階、 得られたｃＤＮＡに、請求項２２〜２５に記載の一本鎖
ＤＮＡ配列を用いてポリメラーゼ連鎖反応を行う段階、
および増幅された非Ａ非Ｂ型肝炎ウイルス遺伝子を検出
する段階、を含む方法。26. A method for detecting a non-A non-B hepatitis virus gene in a sample, comprising the steps of extracting RNA from the sample and synthesizing cDNA by acting a reverse transcriptase on the obtained RNA. Performing a polymerase chain reaction on the obtained cDNA using the single-stranded DNA sequence according to claims 22 to 25,
And detecting the amplified non-A non-B hepatitis virus gene.
抗体を検出するための免疫学的検出方法であつて、 抗非Ａ非Ｂ型肝炎ウイルス抗体を含む疑いのある検体
を、請求項２０に記載の１種以上の組換え非Ａ非Ｂ型肝
炎特異抗原ポリペプチドと一緒に抗原−抗体反応を起こ
す条件下でインキュベーションする段階、および抗原−
抗体複合体を検出する段階、を含む方法。27. An immunological detection method for detecting an antibody against a non-A non-B hepatitis virus antigen, wherein a sample suspected of containing an anti-non-A non-B hepatitis virus antibody is used. Incubating with one or more recombinant non-A non-B hepatitis specific antigen polypeptides as described under conditions that produce an antigen-antibody reaction, and antigen-
Detecting the antibody complex.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3189268A JPH0584085A (en) | 1990-07-09 | 1991-07-03 | Dna fragment encoding non-a non-b type hepatitis specific antigen, its manifestation and method for detecting non-a non-b type hepatitis virus |
| TW080105205A TW268049B (en) | 1990-07-09 | 1991-07-04 |
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2-180889 | 1990-07-09 | ||
| JP18088990 | 1990-07-09 | ||
| JP2-339589 | 1990-11-30 | ||
| JP33958990 | 1990-11-30 | ||
| JP3189268A JPH0584085A (en) | 1990-07-09 | 1991-07-03 | Dna fragment encoding non-a non-b type hepatitis specific antigen, its manifestation and method for detecting non-a non-b type hepatitis virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0584085A true JPH0584085A (en) | 1993-04-06 |
Family
ID=27324920
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3189268A Pending JPH0584085A (en) | 1990-07-09 | 1991-07-03 | Dna fragment encoding non-a non-b type hepatitis specific antigen, its manifestation and method for detecting non-a non-b type hepatitis virus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0584085A (en) |
-
1991
- 1991-07-03 JP JP3189268A patent/JPH0584085A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR0181517B1 (en) | Non-a, non-b hepatitis-specific antigen and its use in hepatitis diagnosis | |
| CA2032381C (en) | Viral agent | |
| JP2693908B2 (en) | NANBV diagnostics | |
| JP3746291B2 (en) | Hepatitis E virus vaccine and inoculation method thereof | |
| WO1991004262A1 (en) | New hcv isolates | |
| EP0476130B1 (en) | Enterically transmitted non-a/non-b hepatitis viral agent and characteristic epitopes thereof | |
| JP2007031440A (en) | Enterically transmitted non-a non-b hepatitis viral factor | |
| US5871904A (en) | Immunassay of non-A, non-B hepatitis virus-related antigens, monoclonal antibodies for use therein, and hybridomas producing the antibodies | |
| WO1994026902A1 (en) | Nucleic acids encoding human astrovirus serotype 2 and uses thereof | |
| EP0586065B1 (en) | Antigenic peptides for grouping hepatitis C virus, kit comprising the same and methods for its grouping using the same | |
| EP0736601A2 (en) | Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use | |
| AU718503B2 (en) | Nucleotide and amino acid sequences of hypervariable region 1 of the envelope 2 gene of hepatitis C virus | |
| US5789559A (en) | DNA sequences of enterically transmitted non-A/non-B hepatitis viral agent | |
| US5824649A (en) | DNA sequences of enterically transmitted non-A/non-B hepatitis viral agent and characteristic epitopes thereof | |
| JPH0584085A (en) | Dna fragment encoding non-a non-b type hepatitis specific antigen, its manifestation and method for detecting non-a non-b type hepatitis virus | |
| JP3195799B2 (en) | DNA fragment encoding non-A non-B hepatitis virus structural protein | |
| WO1992016543A1 (en) | NORWALK VIRUS HUMAN GASTROENTERITIS AGENT AND MOLECULAR CLONING OF CORRESPONDING cDNAs | |
| JPH06507552A (en) | DNA sequences and encoded polypeptides useful in the diagnosis of hepatitis diseases | |
| CA2330935C (en) | A mutant human hepatitis b viral strain and uses thereof | |
| JPH04144686A (en) | Structural protein gene, recombinant vector, escherichia coli transformed therewith, polypeptide and its production | |
| JP3669717B2 (en) | Antigenic peptide for hepatitis C virus grouping, kit containing the same and grouping method using the same | |
| WO1991004341A1 (en) | Hdv-specific peptides | |
| WO1992020803A1 (en) | THE ASTROVIRUS HUMAN GASTROENTERITIS AGENTS AND MOLECULAR CLONING OF CORRESPONDING cDNAs | |
| WO1992018532A1 (en) | Rna, dna and virus antigen protein of non-a non-b hepatitis virus | |
| JPH061799A (en) | Antigen peptide of hepatitis virus of non-a non-b type, nucleic acid fragment encoding the same peptide and its use |