JP3472319B2

JP3472319B2 - Recombinant polypeptide comprising amino acid sequence of non-A non-B hepatitis virus antigen and method for detecting non-A non-B hepatitis virus antibody using said polypeptide

Info

Publication number: JP3472319B2
Application number: JP14794493A
Authority: JP
Inventors: 慎太郎八木; 富子柏熊; 尚子池口
Original assignee: Advanced Life Science Institute Inc
Current assignee: Advanced Life Science Institute Inc
Priority date: 1993-06-18
Filing date: 1993-06-18
Publication date: 2003-12-02
Anticipated expiration: 2018-12-02
Also published as: JPH07133291A

Description

Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】本発明は、抗ＨＣＶ抗体と効率良
く反応する組換え抗原ポリペプチド及び該ポリペプチド
を抗原として用いた非Ａ非Ｂ型肝炎患者血清中の非Ａ非
Ｂ型肝炎ウイルス抗体を効率良く検出する方法に関す
る。TECHNICAL FIELD The present invention relates to a recombinant antigen polypeptide which reacts efficiently with an anti-HCV antibody and a non-A non-B hepatitis virus in the serum of a non-A non-B hepatitis patient using the polypeptide as an antigen. The present invention relates to a method for efficiently detecting an antibody.

【０００２】[0002]

【従来の技術】非Ａ非Ｂ型肝炎は伝染性の肝炎であり、
ウイルスを媒体として伝播すると考えられている。非Ａ
非Ｂ型肝炎の伝播経路は今だ明らかになっていない部分
が多いが、輸血、血液製剤により引き起こされる非Ａ非
Ｂ型肝炎は、Ｂ型肝炎のスクリーニング体制が確立され
た今日、輸血後肝炎として医療上の大きな問題点となっ
ている。Non-A non-B hepatitis is an infectious hepatitis,
It is believed that the virus spreads as a medium. Non-A
Although the transmission route of non-B hepatitis remains largely unknown, non-A non-B hepatitis caused by blood transfusion and blood products has been established today as a screening system for hepatitis B has been established. Has become a major medical problem.

【０００３】１９８９年米国カイロン社のＭ．Ｈｏｕｇ
ｈｔｏｎ等により非Ａ非Ｂ型肝炎に関連したウイルス遺
伝子の一部がクローニングされ（特表平２−５００８８
０号公報）、Ｃ型肝炎ウイルス（ＨＣＶ）と命名され
た。また、これとほぼ同時期に本出願人を含む多くの研
究グループによりＨＣＶ遺伝子が数多く単離され、その
構造上の特徴が明らかとなった。1989 M.K. Houg
A part of the viral gene related to non-A non-B hepatitis was cloned by Hton et al.
No. 0), hepatitis C virus (HCV). At about the same time, a large number of HCV genes were isolated by many research groups including the applicant of the present invention, and their structural features were clarified.

【０００４】推定されるＨＣＶ遺伝子は約９４００塩基
からなる＋鎖のＲＮＡをゲノムとして持ち、約３０００
アミノ酸からなる一つながりのポリペプチドをコードし
ていると考えられる。予想されるアミノ酸配列はフラビ
ウイルス又はペスチウイルスと相同性を持ち、これらの
ウイルスに近縁のウイルスであろうと考えられている。The putative HCV gene has a plus-strand RNA consisting of about 9400 bases as a genome, and has about 3000
It is considered to encode a chain of polypeptides consisting of amino acids. The predicted amino acid sequence shares homology with flaviviruses or pestiviruses and is believed to be a virus closely related to these viruses.

【０００５】これらのウイルス構造との比較から、ＨＣ
Ｖゲノムによってコードされるポリペプチドは、１本の
ポリペプチドとして細胞内において合成された後に、ア
ミノ末端から構造タンパク質であるコア、エンベロープ
（Ｅ１）、及びＮＳ１若しくはＥ２（ＮＳ１／Ｅ２）並
びに非構造タンパク質であるＮＳ２、ＮＳ３、ＮＳ４及
びＮＳ５に切断され、それぞれの機能を果たすと考えら
れている。From comparison with these virus structures, HC
The polypeptide encoded by the V genome is synthesized in the cell as a single polypeptide, and then, from the amino terminus, structural proteins such as core, envelope (E1), and NS1 or E2 (NS1 / E2) It is considered to be cleaved by the proteins NS2, NS3, NS4 and NS5, and fulfill their respective functions.

【０００６】さて、免疫学的にウイルス粒子を検出する
ためには、ウイルス粒子表面を認識する抗体が必要とな
る。この目的を達成するためには、ＨＣＶ粒子を構成し
ている外被蛋白質を免疫して該抗体を作製することにな
るが、ＨＣＶは未だにイン・ビトロ培養系で増殖させる
ことができず、免疫原として使用可能なほど多量に得る
ことは困難な状況にある。In order to detect virus particles immunologically, an antibody that recognizes the surface of virus particles is required. In order to achieve this purpose, the coat protein that constitutes the HCV particles is immunized to produce the antibody, but HCV cannot be propagated in an in vitro culture system, and the immunization is not possible. It is difficult to obtain a large amount that can be used as a raw material.

【０００７】そこで、最近になって、これら構造及び非
構造タンパク質のうち、コア、ＮＳ３、ＮＳ４に相当す
る領域のｃＤＮＡ断片を、大腸菌、酵母等で発現させる
ことが試みられ、それによって組換えタンパク質が効率
良く多量に得ることが出来るようになった。Therefore, recently, among these structural and nonstructural proteins, it has been attempted to express a cDNA fragment of a region corresponding to core, NS3, NS4 in Escherichia coli, yeast, etc. Can be efficiently obtained in large quantities.

【０００８】非Ａ非Ｂ型慢性肝炎患者の血清中に、これ
らの組換えタンパク質（抗原）に対する抗体が認められ
ることから、これらの抗原を用いたＨＣＶ抗体診断薬が
現在広く用いられている。Since antibodies against these recombinant proteins (antigens) are found in the sera of non-A non-B chronic hepatitis patients, HCV antibody diagnostic agents using these antigens are currently widely used.

【０００９】これらの組換え抗原を用いることにより、
非Ａ非Ｂ型慢性肝炎患者のうち約９割に反応する抗体を
検出することが出来る。しかしながら、さらに高い精度
を求めるためには、ＨＣＶの他の領域を抗原として用い
ることが必要である。By using these recombinant antigens,
Antibodies that react with about 90% of non-A non-B chronic hepatitis patients can be detected. However, in order to obtain even higher accuracy, it is necessary to use other regions of HCV as antigens.

【００１０】[0010]

【発明が解決しようとする課題】抗原として利用する領
域は無作為に選択出来るわけではなく、非Ａ非Ｂ型肝炎
患者体内で抗体が産生される領域であることが必要であ
る。The region to be used as an antigen cannot be randomly selected, and it is necessary that it is a region where antibodies are produced in the body of non-A non-B hepatitis patients.

【００１１】さて、ＨＣＶの構造タンパク質のうち、Ｅ
１、ＮＳ１／Ｅ２はＨＣＶの外被タンパク質であると推
定されている。ウイルスの外被タンパク質は強い抗原性
を示すことが多いことから、Ｅ１、ＮＳ１／Ｅ２を抗原
として用いることにより患者血清中の抗体を効率よく検
出できることが期待出来る。Among the structural proteins of HCV, E
1, NS1 / E2 is presumed to be a coat protein of HCV. Since the coat protein of the virus often shows strong antigenicity, it can be expected that the antibody in the patient serum can be efficiently detected by using E1 and NS1 / E2 as the antigen.

【００１２】これらの構造タンパク質のうちＥ１領域は
そのほとんどが疎水的なアミノ酸から構築されている。
疎水的なアミノ酸配列の示す抗原性は一般に低いことか
ら、抗体を検出する抗原としてはあまり適当でないと考
えられる。加えてこのアミノ酸配列を、既知の種々のＨ
ＣＶ間で比較すると、例えば代表的なＨＣＶ株である、
カイロン社から報告されたＨＣＶ−Ｈ株と岡本等によっ
て報告されたＨＣＶ−Ｊ６株を比較すると５０％の相同
性しか示さない。このことから、この領域はＨＣＶ株間
で保存されていないことが分かる。即ち、一つのＨＣＶ
株から単離した抗原を用いて血清中の抗体を検出して
も、その検出効率は低いことが予想できる。[0012] Of these structural proteins, most of the E1 region is constructed from hydrophobic amino acids.
Since the hydrophobic amino acid sequence generally shows low antigenicity, it is considered that it is not suitable as an antigen for detecting an antibody. In addition, the amino acid sequence is
When comparing between CVs, for example, it is a typical HCV strain,
The HCV-H strain reported by Chiron and the HCV-J6 strain reported by Okamoto et al. Show only 50% homology. This indicates that this region is not conserved among HCV strains. That is, one HCV
Even if the antibody in the serum is detected using the antigen isolated from the strain, its detection efficiency can be expected to be low.

【００１３】一方、ＮＳ１／Ｅ２領域については、親水
性のアミノ酸からなる領域及び疎水性のアミノ酸からな
る領域からなり、抗原性を持つ領域を推定すると（Ｊａ
ｍｅｓｏｎＢ．Ａ．＆ＷｏｌｆＨ．、Ｃｏｍｐｕ
ｔ．Ａｐｐｌｉｃ．ｉｎｔｈｅＢｉｏｓｃｉｅｎｃ
ｅ４；１８１−１８６）多くの抗原となり得ることが
推測されるアミノ酸配列を持つ領域が存在することが予
測される。またＥ１領域と比較すると株間の相同性が高
いことから、一つのＨＣＶ株から単離した抗原を用いて
血清中の抗体を検出しても、高い検出効率が期待され
る。On the other hand, the NS1 / E2 region is composed of a region consisting of hydrophilic amino acids and a region consisting of hydrophobic amino acids, and a region having antigenicity is estimated (Ja
meson B. A. & Wolf H. , Compu
t. Application. in the Bioscience
e 4 ; 181-186) It is predicted that there is a region having an amino acid sequence that is presumed to serve as many antigens. Moreover, since the homology between the strains is higher than that in the E1 region, high detection efficiency is expected even when antibodies in serum are detected using an antigen isolated from one HCV strain.

【００１４】この様な観点から、本発明者等は、更に詳
細なアミノ酸配列の解析からＨＣＶ遺伝子領域の特定の
アミノ酸配列を含む抗原を用いることにより非Ａ非Ｂ型
慢性肝炎患者血清中の非Ａ非Ｂ型肝炎ウイルス抗体を効
率良く検出できることを見出し、本発明を完成させた。From this point of view, the present inventors have analyzed the amino acid sequence in more detail, and by using an antigen containing a specific amino acid sequence of the HCV gene region, the present inventors have investigated the non-A non-B chronic hepatitis patient serum. The inventors have found that non-A hepatitis A virus antibody can be efficiently detected and completed the present invention.

【００１５】尚、本明細書中でいう『効率の良い検出』
とは、試験しようとする検体（例えば血清）中の非Ａ非
Ｂ型肝炎ウイルス抗体の検出における精度（感度）だけ
でなく、キャリアー（保菌者）とメモリー（過去の感染
の反映）との区別、異なる菌株由来の抗体をも検出でき
る柔軟な適応性などを意味する。Incidentally, the "efficient detection" referred to in the present specification.
Is not only the accuracy (sensitivity) in the detection of non-A non-B hepatitis virus antibodies in the sample to be tested (eg serum), but also the distinction between carriers (carriers) and memory (reflection of past infections). , Flexible flexibility that can detect antibodies derived from different strains.

【００１６】[0016]

【課題を解決するための手段】即ち、本発明は、ＨＣＶ
のＮＳ１抗原のアミノ酸配列番号３８４から４９５まで
のアミノ酸配列を含むポリペプチド、好ましくは配列番
号１及び２で示されるアミノ酸配列を含むポリペプチド
を提供するものである。更に好ましくは、該ポリペプチ
ドは原核細胞宿主によって発現されたものであり、最も
好ましくは、大腸菌細胞によって発現された糖鎖修飾を
受けていないものである。That is, the present invention is directed to HCV
Of the NS1 antigen of SEQ ID NO: 384 to 495, preferably a polypeptide comprising the amino acid sequences shown in SEQ ID NOs: 1 and 2. More preferably, the polypeptide is expressed by a prokaryotic host, and most preferably is not glycosylated as expressed by E. coli cells.

【００１７】本発明はまた、該ポリペプチドを抗原とし
て用い、非Ａ非Ｂ型肝炎ウイルス抗体を検出する方法を
も提供するものである。The present invention also provides a method for detecting a non-A non-B hepatitis virus antibody using the polypeptide as an antigen.

【００１８】まず、ＨＣＶ遺伝子について説明する。First, the HCV gene will be described.

【００１９】本発明者らは前記領域を得るにあたって、
異なる非Ａ非Ｂ型慢性肝炎患者の血漿中から、従来のも
のと異なり且つ互いに異なる２種類のＨＣＶ遺伝子（＃
４及び＃６）をクローニングした。In obtaining the above-mentioned region, the present inventors have
From the plasma of different non-A non-B chronic hepatitis patients, two types of HCV genes (#
4 and # 6) were cloned.

【００２０】図１には、ＨＣＶ（＃４）遺伝子の５′側
構造を、また図２には、ＨＣＶ（＃６）遺伝子の５′側
構造を、それらの制限酵素部位と共に示した。得られた
ＨＣＶ（＃４）遺伝子は主として、５′末端に約３１０
塩基対の非翻訳領域（以下、ｃｏｒｅＩとも称する）、
約５７０塩基対のコア（ＣＯＲＥ）領域、約５７０塩基
対のエンベロープ（ＥＮＶ）領域、約１２８０塩基対の
ＮＳ１領域、約７２０塩基対のＮＳ２領域、及びＮＳ３
領域の一部から構成されることが判明した。また、ＨＣ
Ｖ（＃６）遺伝子もＨＣＶ（＃４）遺伝子と類似の遺伝
子構造を有していた。FIG. 1 shows the 5'side structure of the HCV (# 4) gene, and FIG. 2 shows the 5'side structure of the HCV (# 6) gene together with their restriction enzyme sites. The obtained HCV (# 4) gene mainly contains about 310 at the 5'end.
A base pair untranslated region (hereinafter, also referred to as coreI),
A core (CORE) region of about 570 base pairs, an envelope (ENV) region of about 570 base pairs, an NS1 region of about 1280 base pairs, an NS2 region of about 720 base pairs, and NS3.
It was found to consist of part of the area. Also, HC
The V (# 6) gene also had a similar gene structure to the HCV (# 4) gene.

【００２１】因みに、これら２種類のＨＣＶ遺伝子の配
列決定に際しては、先ず非Ａ非Ｂ型慢性肝炎患者血漿よ
りＲＮＡを取り出し、これに逆転写酵素を作用させてｃ
ＤＮＡを合成し、２種類のプライマーを用いてポリメラ
ーゼ連鎖反応（ＰＣＲ）を行なってＨＣＶの増幅ＤＮＡ
断片を得、常法に従って該ＤＮＡ断片のクローニングを
行ない、最後にＳａｎｇｅｒ（Science, 214, 1205〜12
10 (1981) ）のジデオキシ鎖終止法を用いて塩基配列を
決定するという手順を採用した。この手法により、５′
非翻訳領域（Ａ）、構造遺伝子コア領域（Ｂ）、構造遺
伝子エンベロープ領域（Ｃ）、及び非構造遺伝子ＮＳ１
／ＮＳ２領域（Ｄ）の４つの領域のＤＮＡ断片を得、そ
れらの各塩基配列を決定した。When sequencing these two types of HCV genes, first, RNA is extracted from the plasma of a non-A non-B chronic hepatitis patient, and a reverse transcriptase is allowed to act on this to c
Amplified DNA of HCV by synthesizing DNA and performing polymerase chain reaction (PCR) using two kinds of primers
A fragment was obtained, and the DNA fragment was cloned according to a conventional method. Finally, Sanger (Science, 214 , 1205-12
10 (1981)) was used to determine the nucleotide sequence using the dideoxy chain termination method. By this method, 5 '
Non-translated region (A), structural gene core region (B), structural gene envelope region (C), and non-structural gene NS1
DNA fragments of four regions of the / NS2 region (D) were obtained, and their respective nucleotide sequences were determined.

【００２２】また、５′非翻訳領域から非構造蛋白質Ｎ
Ｓ２領域までの各クローン化ＤＮＡは、決定された塩基
配列から予想される制限酵素部位を用いて継ぎ込むこと
ができた。これによって、５′非翻訳領域（Ａ）と構造
蛋白質コア領域（Ｂ）の継ぎ込み、非翻訳領域（Ａ）か
ら構造蛋白質ＮＳ１（Ｃ）までの継ぎ込み、５′非翻訳
領域（Ａ）から非構造蛋白質ＮＳ１／ＮＳ２（Ｄ）まで
の継ぎ込みを行ない、対応するＤＮＡ断片を保有するク
ローンＡＢ，ＡＢＣ，ＡＢＣＤを調製することができ
た。In addition, from the 5'untranslated region to the nonstructural protein N
Each cloned DNA up to the S2 region could be spliced in using the restriction enzyme sites predicted from the determined nucleotide sequence. As a result, the 5'untranslated region (A) and the structural protein core region (B) are joined together, and the untranslated region (A) is joined to the structural protein NS1 (C) from the 5'untranslated region (A). The non-structural proteins NS1 / NS2 (D) were ligated to each other, whereby clones AB, ABC and ABCD carrying the corresponding DNA fragments could be prepared.

【００２３】また、ＨＣＶ（＃４）遺伝子由来のクロー
ンＡＢＣＤ（ＣＰ−４と命名）からの約３．５kb ＤＮ
Ａ断片はプラスミドに組み込んだ後、大腸菌ＪＭ１０７
株に移入し、形質転換体（E.coli JM107／CP-4）として
微工研菌寄第１２７８６号として平成４年２月２４日付
で寄託されている。Further, about 3.5 kb DN from the clone ABCD (named CP-4) derived from the HCV (# 4) gene
The A fragment was inserted into a plasmid, and then E. coli JM107
It has been transferred to the strain and has been deposited as a transformant (E. coli JM107 / CP-4) on February 24, 1992 as Microindustrial Research Institute No. 12786.

【００２４】次に、決定された各ＤＮＡ断片の配列をも
とに約3500ヌクレオチドから成るＨＣＶ遺伝子（５′非
翻訳領域／ＣＯＲＥ／ＥＮＶ／ＮＳ１／ＮＳ２／ＮＳ
３）の塩基配列及び推定アミノ酸配列を決定し、ＨＣＶ
（＃４）遺伝子に関して決定された配列を配列番号１と
して、またＨＣＶ（＃６）遺伝子に関して決定された配
列を配列番号２として後記配列表中にそれぞれ示した。Next, based on the determined sequence of each DNA fragment, the HCV gene (5 'untranslated region / CORE / ENV / NS1 / NS2 / NS) consisting of about 3500 nucleotides is used.
The nucleotide sequence and deduced amino acid sequence of 3) were determined, and HCV
The sequence determined for the (# 4) gene is shown as SEQ ID NO: 1, and the sequence determined for the HCV (# 6) gene is shown as SEQ ID NO: 2 in the sequence listing below.

【００２５】＃４及び＃６のＨＣＶ遺伝子構造の特徴は
以下のとおりである。The features of the HCV gene structure of # 4 and # 6 are as follows.

【００２６】（１）ＨＣＶ（＃４）遺伝子断片３４６１ヌクレオチドから成り、翻訳領域はヌクレオチ
ド番号３０７〜３４６１（１０５１アミノ酸）であり、
このうちＣＯＲＥ領域はヌクレオチド番号３０７〜８７
９（１９１アミノ酸）、ＥＮＶ（ＥＮＶ１とも称する）
領域はヌクレオチド番号８８０〜１４５５（１９２アミ
ノ酸）、ＮＳ１（ＥＮＶ２とも称する）領域はヌクレオ
チド番号１４５６〜２７３６（４２７アミノ酸）、ＮＳ
２領域及びＮＳ３領域はヌクレオチド番号２７３７〜３
４６１（２４１アミノ酸）に対応している。(1) The HCV (# 4) gene fragment consists of 3461 nucleotides, and the translation region is nucleotide numbers 307 to 3461 (1051 amino acids),
Of these, the CORE region has nucleotide numbers 307 to 87.
9 (191 amino acids), ENV (also referred to as ENV1)
The region is nucleotide numbers 880 to 1455 (192 amino acids), NS1 (also referred to as ENV2) region is the nucleotide numbers 1456 to 2736 (427 amino acids), NS
2 region and NS3 region are nucleotide numbers 2737-3
It corresponds to 461 (241 amino acids).

【００２７】（２）ＨＣＶ（＃６）遺伝子断片３４０１ヌクレオチドから成り、翻訳領域はヌクレオチ
ド番号３０７〜３４０１（１０３１アミノ酸）であり、
このうちＣＯＲＥ領域はヌクレオチド番号３０７〜８７
９（１９１アミノ酸）、ＥＮＶ（ＥＮＶ１とも称する）
領域はヌクレオチド番号８８０〜１４５５（１９２アミ
ノ酸）、ＮＳ１（ＥＮＶ２とも称する）領域はヌクレオ
チド番号１４５６〜２７３６（４２７アミノ酸）、ＮＳ
２領域及びＮＳ３領域はヌクレオチド番号２７３７〜３
４０１（２２１アミノ酸）に対応している。(2) The HCV (# 6) gene fragment consists of 3401 nucleotides, the translation region is nucleotide numbers 307 to 3401 (1031 amino acids),
Of these, the CORE region has nucleotide numbers 307 to 87.
9 (191 amino acids), ENV (also referred to as ENV1)
The region is nucleotide numbers 880 to 1455 (192 amino acids), NS1 (also referred to as ENV2) region is the nucleotide numbers 1456 to 2736 (427 amino acids), NS
2 region and NS3 region are nucleotide numbers 2737-3
Corresponds to 401 (221 amino acids).

【００２８】以上がＨＣＶ遺伝子についての簡単な説明
である。The above is a brief description of the HCV gene.

【００２９】また、本発明では単一の患者血漿から単離
したＨＣＶ遺伝子である＃４を用いたが、本発明でいう
ＨＣＶ遺伝子とはヒト血漿（又は血清）から単離したも
のであればどのようなものでも発明の範囲に包含される
ものとする。In the present invention, the HCV gene # 4 isolated from the plasma of a single patient was used, but the HCV gene in the present invention means that it is isolated from human plasma (or serum). Anything shall be included in the scope of the invention.

【００３０】前記領域をＨＣＶ遺伝子から単離する方法
は、前記領域を切り出せる適当な制限酵素を用いるなど
の種々の方法が考えられるが、本発明においては、前記
領域の末端の配列を持つプライマーを合成し、ポリメラ
ーゼ連鎖反応（ＰＣＲ）により前記領域を持つＤＮＡ断
片を増幅させることにより得たものを用いた。Various methods can be considered for isolating the region from the HCV gene, such as using an appropriate restriction enzyme capable of cutting out the region. In the present invention, a primer having a sequence at the end of the region is used. Was synthesized and used by amplifying a DNA fragment having the above region by polymerase chain reaction (PCR).

【００３１】前記領域を組換えタンパク質として発現さ
せるためには、種々の宿主を用いて発現させることが可
能である。例えば酵母、大腸菌、ワクチニアウイルス、
バキュロウイルス、哺乳類細胞などが例示できる。In order to express the above region as a recombinant protein, it can be expressed using various hosts. For example, yeast, E. coli, vaccinia virus,
Examples include baculovirus and mammalian cells.

【００３２】該領域にはＮ結合型の糖鎖結合部位と予測
されるアミノ酸の配列が存在し、該領域を糖鎖修飾が行
なわれる宿主、すなわち真核細胞を宿主とし、細胞内分
泌経路にのせるための適当なシグナル配列を用いて発現
させることにより、糖鎖修飾を受けた組換えタンパク質
を生産することが可能となる。A sequence of amino acids predicted to be an N-linked sugar chain-binding site exists in the region, and the region is used as a host for sugar chain modification, that is, a eukaryotic cell as a host, to By using an appropriate signal sequence for expression, expression of a sugar chain-modified recombinant protein can be produced.

【００３３】しかしながらこのような方法で生産させた
組換えタンパク質は、例えば著しい糖鎖修飾を受けるこ
とにより、糖鎖が抗原抗体反応を阻害するなどの影響を
示し、結果として検査しようとする血清中の抗体の検出
率が低下するなどの悪影響が懸念される。従って、該領
域を組換えタンパク質として発現させるためには、この
ような修飾を受けない形で作らせることが望ましい。However, the recombinant protein produced by such a method shows an effect that the sugar chain inhibits the antigen-antibody reaction, for example, by undergoing remarkable sugar chain modification, and as a result, in the serum to be tested. There is concern about adverse effects such as a decrease in the antibody detection rate of. Therefore, in order to express the region as a recombinant protein, it is desirable that the region is not modified.

【００３４】そこで、本発明においては糖鎖修飾を受け
ない形で組換えタンパク質を得るために、大腸菌を宿主
として生産させる方法を選択した。Therefore, in the present invention, a method of producing Escherichia coli as a host was selected in order to obtain a recombinant protein in a form not subjected to sugar chain modification.

【００３５】プロモーターとしては発現させるのに適当
なプロモーターを持つベクターであればどのようなもの
でも良く、例えばトリプトファン合成酵素オペロン、ラ
クトースオペロン、λファージＰ_LＰ_R、Ｔ７ＲＮＡポ
リメラーゼ等を用いることが出来る。The promoter may be any vector as long as it has a suitable promoter for expression, and for example, tryptophan synthase operon, lactose operon, λ phage P _L P _R , T7 RNA polymerase and the like can be used. .

【００３６】本発明においてはトリプトファン合成酵素
オペロンをプロモーターとして持つベクターを用いて該
領域をＴｒｐＥとの融合タンパク質として発現させるこ
とにより、効率良く組換えタンパク質を得ることが出来
ることを示したが、方法としてはこれに限定されるもの
ではない。In the present invention, it was shown that a recombinant protein can be efficiently obtained by expressing this region as a fusion protein with TrpE using a vector having a tryptophan synthase operon as a promoter. However, the present invention is not limited to this.

【００３７】前記のごとく発現させた組換えタンパク質
は、菌体を粉砕し、可溶化させたのち、ゲル濾過等のク
ロマトグラフィーにより単離精製することが可能であ
る。The recombinant protein expressed as described above can be isolated and purified by chromatography such as gel filtration after crushing the cells and solubilizing the cells.

【００３８】精製させたタンパク質は酵素抗体法により
非Ａ非Ｂ型慢性肝炎患者血清中の抗体を効率良く検出で
きた（表１）。このことからも該領域を含む組換えタン
パク質抗原は、非Ａ非Ｂ型慢性肝炎患者血清中の抗体検
出に有用なことは明らかである。The purified protein was able to efficiently detect the antibody in the serum of the non-A non-B chronic hepatitis patient by the enzyme antibody method (Table 1). From this, it is clear that the recombinant protein antigen containing the region is useful for detecting the antibody in the serum of non-A non-B chronic hepatitis patients.

【００３９】この様に糖鎖修飾を受けない形で得た組換
えタンパク質が、非Ａ非Ｂ型慢性肝炎患者血清中の抗体
を効率良く検出できることから、該領域のアミノ酸配列
からなる化学合成したポリペプチドを用いることにより
抗体を検出できることも、当業者には容易に類推できる
ことである。Since the recombinant protein thus obtained in the form not subjected to sugar chain modification can efficiently detect the antibody in the serum of the non-A non-B chronic hepatitis patient, it was chemically synthesized from the amino acid sequence of the region. The fact that an antibody can be detected by using a polypeptide can be easily inferred by those skilled in the art.

【００４０】以下実施例によって本発明をさらに詳細に
説明するが、本発明はこの範囲に限定されるものでな
い。The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to this range.

【００４１】[0041]

【Example】

実施例１組換え抗原発現プラスミドの作成ＮＳ１領域は、非Ａ非Ｂ型慢性肝炎患者血漿より取り出
したＲＮＡを鋳型に作製したｃＤＮＡを用い、２種類の
プライマーを用いてポリメラーゼ連鎖反応（ＰＣＲ）を
行なわせることによって増幅されたＨＣＶ遺伝子断片を
連結させた、前述のプラスミドＣＰ−４から得た。Example 1 Preparation of Recombinant Antigen Expression Plasmid For the NS1 region, cDNA prepared using RNA extracted from plasma of a non-A non-B chronic hepatitis patient as a template was used, and polymerase chain reaction (PCR) was performed using two kinds of primers. It was obtained from the above-mentioned plasmid CP-4, to which the HCV gene fragment amplified by carrying out was ligated.

【００４２】ＮＳ１領域を前記プラスミドより取り出す
ために、下記の配列を持つプライマー２種類を合成し
た。In order to extract the NS1 region from the above plasmid, two kinds of primers having the following sequences were synthesized.

【００４３】ＮＳ１−Ｈｉｎｄ；ＴＴＴＴＡＡＧＣＴＴ
ＧＧＧＧＧＡＣＣＣＡＣＧＴＧＴＣＧＧＧＧＮＳ１−２；ＡＡＡＧＧＡＴＣＣＴＡＣＣＧＣＡＣＧＧ
ＣＣＣＧＡＧＧＣＧＣこれらのプライマーを用いて以下
の条件でＰＣＲを行なわせた。１ｎｇのＣＰ−４のＤＮ
Ａを鋳型に５０ｐｍｏｌＮＳ１−Ｈｉｎｄプライマー、
５０ｐｍｏｌＮＳ１−２プライマー、６７ｍＭＴｒｉ
ｓ−ＨＣｌ（ｐＨ８．８）、１６．６ｍＭ（ＮＨ₄）
₂ＳＯ₄、０．２ｍＭｄＮＴＰ、２ｍＭＭｇＣ
ｌ₂、０．２ｍｇ／ｍｌゼラチン、０．０５％Ｔｒｉ
ｔｏｎＸ−１００となるよう反応液を混合し（全反応液
５０μｌ）、２．５単位のＴａｑＤＮＡポリメラーゼを
加え、パラフィンオイルを重層させた後、９４℃、３０
秒、５５℃、６０秒、７２℃、１２０秒の条件で３０サ
イクル反応を行なった。パラフィンオイルを除去した
後、水層をクロロホルムにより抽出し、続いてエタノー
ル沈殿によりＤＮＡ回収した。回収したＤＮＡを４０μ
ｌのＴＥ溶液に溶解させた後、１０μｌを分取し、Ｅｃ
ｏＲＩ反応液（０．５ＭＴｒｉｓ−ＨＣｌ［ｐＨ７．
５］、０．５ＭＮａＣｌ、７０ｍＭＭｇＣｌ₂）を
１．５μｌ加え、ここにＨｉｎｄIII 、ＢａｍＨＩをそ
れぞれ１μｌずつ加え３７℃で１時間反応させた。この
反応液をアガロース電気泳動にかけ約２００ｂｐの断片
を分離した。分離した断片をガラスパウダー法（Ｇｅｎ
ｅＣｌｅａｎII、ＢＩＯ１０１社）により５μｌのＴ
Ｅ溶液に回収した。回収したＤＮＡ溶液に１μｌのＨｉ
ｎｄIII とＢａｍＨＩで切断したｐＵＣ１１９プラスミ
ド溶液（１０ｎｇ／μｌ）を加え、ｌｉｇａｔｉｏｎＡ
液３０μｌとｌｉｇａｔｉｏｎＢ液５μｌ（ＤＮＡラ
イゲーションキット、宝酒造）を加え、充分に混合し、
１６℃で１時間反応させた。得られたＤＮＡ溶液１０μ
ｌを用いて大腸菌ＸＬ１−ｂｌｕｅ（Ｓｔｒａｔａｇｅ
ｎｅ社）Ｈａｎａｈａｎの方法［ＤＮＡｃｌｏｎｉ
ｎｇ：Ａｐｒａｃｔｉｃａｌａｐｐｒｏａｃｈ（ｅ
ｄ．Ｄ．Ｍ．Ｇｌｏｖｅｒ）、ｖｏｌ．１、ｐ１０９
〜、ＩＲＣｐｒｅｓｓ、（１９８５）］に従って形質
転換させ、Ｘ−Ｇａｌ、ＩＰＴＧを含むＬ−ａｍｐプレ
ートに蒔き、白色のコロニーを選択することによりアン
ピシリン耐性で、βガラクトシダーゼ欠損のコロニーを
選択した。NS1-Hind; TTTTAAGCTT
GGGGGACCCACGTGTCGGGGG NS1-2; AAAGGATCCTACCCGCACGG
CCCGAGGCGC PCR was performed using these primers under the following conditions. 1 ng CP-4 DN
50 pmol NS1-Hind primer using A as a template,
50 pmol NS1-2 primer, 67 mM Tri
s-HCl (pH8.8), 16.6mM (NH 4)
₂ SO ₄ , 0.2 mM dNTP, 2 mM MgC
l ₂ , 0.2 mg / ml gelatin, 0.05% Tri
The reaction solution was mixed to give tonX-100 (total reaction solution 50 μl), 2.5 units of Taq DNA polymerase was added, paraffin oil was overlaid, and then 94 ° C., 30
The reaction was performed for 30 cycles under the conditions of seconds, 55 ° C., 60 seconds, 72 ° C. and 120 seconds. After removing the paraffin oil, the aqueous layer was extracted with chloroform, and then DNA was recovered by ethanol precipitation. 40μ of recovered DNA
After dissolving in 1 l TE solution, 10 μl aliquot
oRI reaction solution (0.5 M Tris-HCl [pH 7.
5], 1.5 μl of 0.5 M NaCl, 70 mM MgCl ₂ ) was added, and 1 μl each of HindIII and BamHI was added thereto, and the mixture was reacted at 37 ° C. for 1 hour. This reaction solution was subjected to agarose electrophoresis to separate a fragment of about 200 bp. The separated fragments are processed by the glass powder method (Gen
e CleanII, BIO101 company) 5 μl T
Collected in E solution. 1 μl of Hi was added to the recovered DNA solution.
A pUC119 plasmid solution (10 ng / μl) cleaved with ndIII and BamHI was added, and ligationA was added.
Add 30 μl of the solution and 5 μl of the ligation B solution (DNA Ligation Kit, Takara Shuzo) and mix thoroughly.
The reaction was carried out at 16 ° C for 1 hour. Obtained DNA solution 10μ
E. coli XL1-blue (Stratage)
ne company) Method of Hanahan [DNA clone
ng: A practical approach (e
d. D. M. Glover), vol. 1, p109
, IRC press, (1985)], seeded on L-amp plates containing X-Gal and IPTG, and selected white colonies to select ampicillin-resistant β-galactosidase-deficient colonies.

【００４４】形質転換体のＤＮＡをミニプレパレーショ
ン法により調製し、制限酵素で切断し、目的の大きさの
断片が挿入されているプラスミドを持つ形質転換体を選
択した。この形質転換体から、プラスミドＤＮＡを調製
し、調製した組換え体プラスミドＤＮＡ約１μｇをＭ１
３ＲＰ１プライマー（アプライドバイオシステム社）又
は−２１Ｍ１３プライマー（アプライドバイオシステム
社）を用い、Ｃｙｃｌｉｎｇｓｅｑｕｅｎｃｉｎｇ法
（アプライドバイオシステム社テクニカルマニュアル）
によりメーカーの推奨する方法に従い反応させ、反応産
物をＤＮＡｓｅｑｕｅｎｃｅｒＭｏｄｅｌ３７０Ａ
（ｖｅｒｓｉｏｎ１．３０、アプライドバイオシステ
ム社）を用いて分析し塩基配列を決定した。The DNA of the transformant was prepared by the mini-preparation method, cut with a restriction enzyme, and a transformant having a plasmid in which a fragment of the desired size was inserted was selected. Plasmid DNA was prepared from this transformant, and about 1 μg of the prepared recombinant plasmid DNA was added to M1.
Cycle sequencing method (Applied Biosystems technical manual) using 3RP1 primer (Applied Biosystems) or -21M13 primer (Applied Biosystems)
According to the method recommended by the manufacturer, and the reaction product is the DNAsequencer Model 370A.
(Version 1.30, Applied Biosystems) was used to analyze and determine the nucleotide sequence.

【００４５】このようにしてＮＳ１領域ＤＮＡ断片を持
つプラスミドｐＮＳ＃２を得た。決定した塩基配列から
予定の翻訳停止コドンが挿入されていないことが明らか
になったため、翻訳停止コドンをプラスミドに挿入する
こととした。翻訳停止コドンは以下の方法により該プラ
スミドに挿入した。プラスミドｐＮＳ＃２１μｇをＢ
ａｍＨＩで切断した（１０ｍＭＴｒｉｓ−ＨＣｌ［ｐ
Ｈ７．５］、７ｍＭＭｇＣｌ₂、１５０ｍＭＮａＣ
ｌ、１０ｕｎｉｔｓＢａｍＨＩ／反応液量１０μｌで
３７℃１時間反応）後、８０μｌのＴＥ溶液、１０μｌ
の１ＭＴｒｉｓ−ＨＣｌ（ｐＨ８．５）を加え、１μ
ｌのＢａｃｔｅｒｉａｌＡｌｋａｌｉｎｅＰｈｏｓ
ｐｈａｔａｓｅ（１２Ｕ／μｌ）を加え６５℃で１時間
反応させた。反応終了後ガラスパウダー法（Ｇｅｎｅ
ＣｌｅａｎII、ＢＩＯ１０１社）によりＤＮＡを１０μ
ｌのＴＥ溶液に回収した。ＤＮＡｂｌｕｎｔｉｎｇ
ｋｉｔ（宝酒造）を用いメーカーの推奨する方法により
ＢａｍＨＩ切断末端を平滑末端化させた。反応終了後フ
ェノール／クロロホルム混合液により溶液を抽出した
後、エタノール沈殿法によりＤＮＡを回収した。回収し
たＤＮＡを１０μｌのＴＥ溶液に溶解した後、１μｌを
用い１μｌのユニバーサルトランスレーショナルターミ
ネーター（Ｐｈａｒｍａｃｉａ社）と共にライゲーショ
ン反応を行なわせた（５０ｍＭＴｒｉｓ−ＨＣｌ［ｐ
Ｈ７．５］、１０ｍＭＭｇＣｌ₂、１０ｍＭＤＴ
Ｔ、１ｍＭＡＴＰ、３５０ｕｎｉｔｓＴ４ＤＮＡ
ｌｉｇａｓｅ／反応液量１０μｌで１６℃１２時間反
応）。反応液量を６５℃１０分間処理した後、１ＭＮ
ａＣｌを１μｌ、ＢａｍＨＩ、ＣｌａＩをそれぞれ１μ
ｌ加え、３７℃１時間反応させた。反応液を６５℃で１
０分間処理した後、その１０μｌを用いて大腸菌ＸＬ１
−ｂｌｕｅ（Ｓｔｒａｔａｇｅｎｅ社）をＨａｎａｈａ
ｎの方法に従って形質転換した。アンピシリン耐性のコ
ロニーを選択し、形質転換体のＤＮＡをミニプレパレー
ション法により調製し、制限酵素で切断し、ユニバーサ
ルトランスレーショナルターミネーター断片が挿入され
ているプラスミドを持つ形質転換体を選択した。このプ
ラスミドをｐＮＳ＃２−４と命名した。Thus, the plasmid pNS # 2 having the NS1 region DNA fragment was obtained. Since it was revealed from the determined nucleotide sequence that the intended translation stop codon was not inserted, it was decided to insert the translation stop codon into the plasmid. The translation stop codon was inserted into the plasmid by the following method. Plasmid pNS # 2 1 μg was added to B
cleaved with amHI (10 mM Tris-HCl [p
H7.5], 7 mM MgCl ₂ , 150 mM NaC
1, 10units BamHI / reaction liquid volume 10 μl, reacted at 37 ° C. for 1 hour), and then 80 μl TE solution, 10 μl
1M Tris-HCl (pH 8.5) was added to 1 μm.
l Bacterial Alkaline Phos
Phasease (12 U / μl) was added and the mixture was reacted at 65 ° C. for 1 hour. After completion of reaction, glass powder method (Gene
CleanII, BIO101 company) DNA 10μ
1 of TE solution. DNA blunting
The BamHI-cut ends were made blunt by the method recommended by the manufacturer using kit (Takara Shuzo). After completion of the reaction, the solution was extracted with a phenol / chloroform mixed solution, and then DNA was recovered by the ethanol precipitation method. The recovered DNA was dissolved in 10 μl of TE solution, and then 1 μl was used to carry out a ligation reaction with 1 μl of a universal translation terminator (Pharmacia) (50 mM Tris-HCl [p.
H7.5], 10 mM MgCl ₂ , 10 mM DT
T, 1 mM ATP, 350 units T4 DNA
ligase / reaction solution volume of 10 μl, reacted at 16 ° C. for 12 hours) After treating the reaction solution volume at 65 ° C for 10 minutes, 1M N
aCl 1 μl, BamHI, ClaI 1 μl
1 was added and reacted at 37 ° C. for 1 hour. Reaction mixture at 65 ℃ 1
After treatment for 0 minutes, 10 μl of the treated E. coli XL1 was used.
-Blue (Stratagene) to Hanaha
Transformation was performed according to the method of n. Ampicillin-resistant colonies were selected, the DNA of the transformant was prepared by the mini-preparation method, cut with a restriction enzyme, and a transformant having a plasmid into which the universal translation terminator fragment was inserted was selected. This plasmid was designated as pNS # 2-4.

【００４６】ｐＮＳ＃２−４１μｇをＳｍａＩ、Ｈｉ
ｎｄIII で切断した（１０ｍＭＴｒｉｓ−ＨＣｌ［ｐ
Ｈ７．５］、７ｍＭＭｇＣｌ₂、２０ｍＭＫＣｌ、
１０ｕｎｉｔｓＨｉｎｄIII 、１０ｕｎｉｔｓＳｍ
ａＩ／反応液量１０μｌで３７℃１時間反応）後、この
反応液をアガロース電気泳動にかけ約２００ｂｐの断片
を分離した。分離した断片をガラスパウダー法により５
μｌのＴＥ溶液に回収した。一方ｐＡｔ−Ｔｒｐ−Ｔｒ
ｐＥ−Ｃ１４−２（特願平４−２１２０６１）１μｇを
ＥｃｏＲＩで切断した（５０ｍＭＴｒｉｓ−ＨＣｌ
［ｐＨ７．５］、７ｍＭＭｇＣｌ₂、５０ｍＭＮａ
Ｃｌ、１０ｕｎｉｔｓＥｃｏＲＩ／反応液量１０μｌ
で３７℃、１時間反応）後、反応液を６５℃で１０分間
処理し、１ＭＮａＣｌを１μｌ、ＳａｌＩを１μｌ加
え、３７℃で１時間反応させた。この反応液をアガロー
ス電気泳動にかけ約３Ｋｂｐの断片を分離した。分離し
た断片をガラスパウダー法により１０μｌのＴＥ溶液に
回収した。この回収したＤＮＡ溶液１μｌと先の回収し
た約２００ｂｐの断片５μｌを混合し、ｌｉｇａｔｉｏ
ｎＡ液３０μｌとｌｉｇａｔｉｏｎＢ液５μｌ（Ｄ
ＮＡライゲーションキット、宝酒造）を加え、良く混合
した後１６℃で１時間反応させた。この溶液１０μｌを
用いて大腸菌ＸＬ１−ｂｌｕｅ（Ｓｔｒａｔａｇｅｎｅ
社）をＨａｎａｈａｎの方法に従って形質転換した。
アンピシリン耐性のコロニーを選択し、形質転換体のＤ
ＮＡをミニプレパレーション法により調製し、制限酵素
で切断することにより両断片が環状化しているプラスミ
ドを持つ形質転換体を選択した。この組換え抗原発現プ
ラスミドをｐＴｒｐＥ−ＮＳ＃２と命名した。1 μg of pNS # 2-4 was added to SmaI and Hi.
cleaved with ndIII (10 mM Tris-HCl [p
H7.5], 7 mM MgCl ₂ , 20 mM KCl,
10units HindIII, 10units Sm
After reacting with aI / reaction solution volume of 10 μl for 1 hour at 37 ° C., the reaction solution was subjected to agarose electrophoresis to separate a fragment of about 200 bp. 5 pieces of the separated pieces by the glass powder method
It was collected in μl of TE solution. On the other hand, pAt-Trp-Tr
1 μg of pE-C14-2 (Japanese Patent Application No. 4-212061) was digested with EcoRI (50 mM Tris-HCl).
[PH 7.5], 7 mM MgCl ₂ , 50 mM Na
Cl, 10 units EcoRI / reaction volume 10 μl
At 37 ° C. for 1 hour), the reaction solution was treated at 65 ° C. for 10 minutes, 1 μl of 1M NaCl and 1 μl of SalI were added, and the mixture was reacted at 37 ° C. for 1 hour. This reaction solution was subjected to agarose electrophoresis to separate a fragment of about 3 Kbp. The separated fragments were collected in a 10 μl TE solution by the glass powder method. 1 μl of the recovered DNA solution was mixed with 5 μl of the previously recovered fragment of about 200 bp, and the mixture was ligated.
n A solution 30 μl and ligation B solution 5 μl (D
NA ligation kit, Takara Shuzo) was added, mixed well, and reacted at 16 ° C. for 1 hour. E. coli XL1-blue (Stratagene) was prepared using 10 μl of this solution.
Company) was transformed according to the method of Hanahan.
Ampicillin-resistant colonies were selected and the transformant D
NA was prepared by the mini-preparation method and cut with a restriction enzyme to select a transformant having a plasmid in which both fragments were circularized. This recombinant antigen expression plasmid was designated as pTrpE-NS # 2.

【００４７】実施例２組換え抗原の発現と精製ｐＴｒｐＥ−ＮＳ＃２を用いて大腸菌Ｃ６００を形質転
換しアンピシリン耐性株を得た。得られた株を５０μｇ
／ｍｌの濃度のアンピシリンを含むＬ培地４００ｍｌ中
に３７℃において１夜振盪培養した。遠心により菌体を
集め、４ｌのＭ９−ＣＡ培地に再懸濁し、３７℃におい
て１夜振盪培養した。遠心により菌体を集め、５０ｍｌ
のＬｙｓｉｓ液［５０ｍＭＴｒｉｓ−ＨＣｌ（ｐＨ
８．５）、３０ｍＭＮａＣｌ、５ｍＭＥＤＴＡ］に
再懸濁し、１ｍｌのリゾチーム液（１０ｍｇ／ｍｌＬ
ｙｓｏｚｙｍｅ）を加え、３７℃において１時間処理し
た。この懸濁液を超音波処理（１５０Ｗ、９０秒で２
回）にかけることにより細胞を破壊した。１５０００ｒ
ｐｍで、４℃において３０分間遠心し不溶性分画を回収
した。不溶性分画を５０ｍｌのＡ溶液［５０ｍＭＴｒ
ｉｓ−ＨＣｌ（ｐＨ８．５）、１％ＮＰ４０］に再懸濁
しホモジナイズ（１５００ｒｐｍで５ストローク）し
た。懸濁液を１５０００ｒｐｍで、４℃において３０分
間遠心し不溶性分画を回収した。不溶性分画を５０ｍｌ
の２Ｍ尿素を含むＡ溶液に再懸濁しホモジナイズ（１５
００ｒｐｍで５ストローク）した。懸濁液を１５０００
ｒｐｍで、４℃において３０分間遠心し不溶性分画を回
収した。不溶性分画を５０ｍｌの６Ｍ尿素を含むＡ溶液
に再懸濁しホモジナイズ（１５００ｒｐｍで５ストロー
ク）した。懸濁液を１５０００ｒｐｍで、４℃において
３０分間遠心し可溶性分画を回収した。Example 2 Expression and Purification of Recombinant Antigen Escherichia coli C600 was transformed with pTrpE-NS # 2 to obtain an ampicillin resistant strain. 50 μg of the obtained strain
The cells were shake-cultured overnight at 37 ° C. in 400 ml of L medium containing ampicillin at a concentration of / ml. The cells were collected by centrifugation, resuspended in 4 l of M9-CA medium, and cultured at 37 ° C with shaking overnight. Collect the cells by centrifugation, 50 ml
Lysis solution [50 mM Tris-HCl (pH
8.5), 30 mM NaCl, 5 mM EDTA] and resuspended in 1 ml lysozyme solution (10 mg / ml L
(ysozyme) was added and treated at 37 ° C. for 1 hour. Sonicate this suspension (150 W, 90 seconds for 2
Cells) to destroy the cells. 15000r
The insoluble fraction was collected by centrifugation at pm for 30 minutes at 4 ° C. The insoluble fraction was added to 50 ml of A solution [50 mM Tr
It was resuspended in is-HCl (pH 8.5), 1% NP40] and homogenized (5 strokes at 1500 rpm). The suspension was centrifuged at 15000 rpm for 30 minutes at 4 ° C. to collect the insoluble fraction. 50 ml of insoluble fraction
Resuspend in A solution containing 2M urea and homogenize (15
5 strokes at 00 rpm). 15,000 suspension
The insoluble fraction was collected by centrifugation at 4 ° C. for 30 minutes at 4 ° C. The insoluble fraction was resuspended in 50 ml of A solution containing 6M urea and homogenized (5 strokes at 1500 rpm). The suspension was centrifuged at 15000 rpm for 30 minutes at 4 ° C. to collect the soluble fraction.

【００４８】ここにジチオスレイトール、塩化ナトリウ
ムをそれぞれ終濃度５０ｍＭ、２００ｍＭとなるように
加え１夜４℃に静置。この溶液を６回に分けて６Ｍ尿素
と５ｍＭのジチオスレイトールを含むＡ溶液で平衡化し
たＳｕｐｅｒｄｅｘ７５ｐｇカラム（２．６ｃｍＸ
１２０ｃｍ）にかけ、毎分２ｍｌの流速で溶出し、４ｍ
ｌずつの分画を採取した。組換えＮＳ１タンパク質を含
む分画を集め、６Ｍ尿素を含むＡ溶液に対し透析を行な
い、ジチオスレイトールを除きＥＬＩＳＡによる抗体検
出用の抗原とした。Dithiothreitol and sodium chloride were added thereto so that the final concentrations were 50 mM and 200 mM, respectively, and the mixture was allowed to stand at 4 ° C. overnight. This solution was divided into 6 times and equilibrated with a solution A containing 6 M urea and 5 mM dithiothreitol, a Superdex 75 pg column (2.6 cm X
120 cm) and elute at a flow rate of 2 ml / min, 4 m
Fractions of 1 were collected. Fractions containing the recombinant NS1 protein were collected and dialyzed against A solution containing 6M urea to remove dithiothreitol and used as an antigen for antibody detection by ELISA.

【００４９】実施例３酵素抗体法による非Ａ非Ｂ型慢
性肝炎患者血清中の抗体の検出精製した抗原を２．５μｇ／ｍｌの濃度となるように８
Ｍ尿素を含む０．１Ｍのリン酸緩衝液（ｐＨ７．５）に
希釈した。希釈した抗原をヌンク社のマルチモジュール
プレートにウェル当たり１００μｌのせ、室温に２時間
静置した。抗原希釈液を除いた後、ウェル当たり１００
μｌのブロッキング液［０．５％ガゼイン、０．１５Ｍ
ＮａＣｌ、２．５ｍＭＥＤＴＡ、０．１Ｍリン酸緩
衝液（ｐＨ７．０）］を加え室温に２時間静置した。ウ
ェルを０．１％Ｔｗｅｅｎ２０を含むＰＢＳで洗浄した
後、検体希釈液［０．５％ガゼイン、０．５ＭＮａＣ
ｌ、２．５ｍＭＥＤＴＡ、０．１Ｍリン酸緩衝液（ｐ
Ｈ７．０）］によって１１倍に希釈した非Ａ非Ｂ型慢性
肝炎患者血清１００μｌを加え４５分間静置した。ウェ
ルを０．１％Ｔｗｅｅｎ２０を含むＰＢＳで洗浄した
後、１００μｌのホースラディシュペルオキシダーゼに
よって標識された抗ヒトＩｇＧ抗体を加え、４５分間静
置した。ウェルを０．１％Ｔｗｅｅｎ２０を含むＰＢ
Ｓで洗浄した後、常法に従い、発色法により酵素活性を
測定した。その結果を表１にまとめて示す。Example 3 Detection of antibody in serum of non-A non-B chronic hepatitis patient by enzyme antibody method Purified antigen was adjusted to a concentration of 2.5 μg / ml.
It was diluted in 0.1 M phosphate buffer (pH 7.5) containing M urea. 100 μl of the diluted antigen was placed in a Nunc multi-module plate per well and allowed to stand at room temperature for 2 hours. 100 per well after removing the antigen diluent
μl blocking solution [0.5% casein, 0.15M
NaCl, 2.5 mM EDTA, 0.1 M phosphate buffer (pH 7.0)] was added and the mixture was allowed to stand at room temperature for 2 hours. The wells were washed with PBS containing 0.1% Tween 20, and then diluted with a sample [0.5% casein, 0.5M NaC].
1, 2.5 mM EDTA, 0.1 M phosphate buffer (p
H 7.0)] was added 11-fold diluted with 100 μl of non-A non-B chronic hepatitis patient serum, and the mixture was allowed to stand for 45 minutes. After washing the wells with PBS containing 0.1% Tween 20, 100 μl of an anti-human IgG antibody labeled with horseradish peroxidase was added, and the mixture was allowed to stand for 45 minutes. Wells with PB containing 0.1% Tween 20
After washing with S, the enzyme activity was measured by a coloring method according to a conventional method. The results are summarized in Table 1.

【００５０】[0050]

【表１】 [Table 1]

【００５１】表中＋は抗体が検出できたことを示し、そ
の数は強度を示す。酵素活性については、６００ｎｍを
リファレンスとして、４９５ｎｍの吸光度を測定した。
１７検体の非Ａ非Ｂ型慢性肝炎患者血清中、１５検体の
患者血清において組換え抗原と反応する抗体を検出でき
た。In the table, + indicates that the antibody could be detected, and the number indicates the intensity. Regarding the enzyme activity, the absorbance at 495 nm was measured with 600 nm as a reference.
In 17 sera of non-A non-B chronic hepatitis patients, 15 sera of patients were able to detect an antibody reactive with the recombinant antigen.

【００５２】[0052]

[Sequence list]

配列番号：１配列の長さ：3461 配列の型：核酸鎖の数：一本鎖トポロジー：直鎖状配列の種類：cDNA to genomic RNA フラグメント型：N 末端フラグメント配列： ATCACTCCCC TGTGAGGAAC TACTGTCTTC ACGCAGAAAG CGTCTAGCCA TGGCGTTAGT 60 ATGAGTGTCG TGCAGCCTCC AGGACCCCCC CTCCCGGGAG AGCCATAGTG GTCTGCGGAA 120 CCGGTGAGTA CACCGGAATT GCCAGGACGA CCGGGTCCTT TCTTGGATTA ACCCGCTCAA 180 TGCCTGGAGA TTTGGGCGTG CCCCCGCGAG ACTGCTAGCC GAGTAGTGTT GGGTCGCGAA 240 AGGCCTTGTG GTACTGCCTG ATAGGGTGCT TGCGAGTGCC CCGGGAGGTC TCGTAGACCG 300 TGCATC ATG AGC ACA AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC 348 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn 1 5 10 ACC AAC CGC CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT GGT CAG ATC 396 Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile 15 20 25 30 GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG 444 Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val 35 40 45 CGC GCG ACT AGG AAG ACT TCC GAG CGG TCG CAA CCT CGT GGA AGG CGA 492 Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg 50 55 60 CAA CCT ATC CCC AAG GCT CGC CGG CCC GAG GGC AGG GCC TGG GCT CAG 540 Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln 65 70 75 CCC GGG TAC CCT TGG CCC CTC TAT GGT AAC GAG GGC CTG GGG TGG GCA 588 Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala 80 85 90 GGA TGG CTC CTG TCA CCC CGC GGC TCC CGG CCT AGT TGG GGC CCC ACG 636 Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr 95 100 105 110 GAC CCC CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC CTC 684 Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu 115 120 125 ACA TGC GGC TTC GCC GAC CTC ATG GGG TAC ATT CCG CTC GTC GGC GCC 732 Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala 130 135 140 CCC CTA GGA GGC GTT GCC AGG GCC CTG GCG CAT GGC GTC CGG GTT CTG 780 Pro Leu Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu 145 150 155 GAA GAC GGC GTG AAT TAC GCA ACA GGG AAT CTG CCC GGT TGC TCT TTC 828 Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe 160 165 170 TCT ATC TTC CTC TTG GCT TTG CTG TCC TGT CTG ACC ATC CCA GCT TCC 876 Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser 175 180 185 190 GCT TAT GAA GTG CGC AAC GTG TCC GGG GTG TAC CAT GTC ACA AAC GAC 924 Ala Tyr Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp 195 200 205 TGC TCC AAC TCA AGT ATT GTG TAT GAG GCA GCG GAC GTG ATC ATG CAC 972 Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val Ile Met His 210 215 220 ACC CCC GGG TGC GTG CCC TGC GTT CGG GAG AGC AAT TTC TCC CGC TGC 1020 Thr Pro Gly Cys Val Pro Cys Val Arg Glu Ser Asn Phe Ser Arg Cys 225 230 235 TGG GTA GCG CTC ACT CCC ACG CTC GCG GCC AGA AAC AGC AGC ATC CCC 1068 Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ser Ser Ile Pro 240 245 250 ACT ACG ACA ATA CGA CGC CAT GTC GAT TTG CTC GTT GGG GCA GCT GCT 1116 Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala 255 260 265 270 CTC TGC TCC GCC ATG TAC GTG GGG GAT CTC TGC GGA TCT GTC TTC CTC 1164 Leu Cys Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu 275 280 285 GTC TCC CAG CTG TTC ACC TTC TCA CCT CGC CGG TAT GAG ACG GTA CAG 1212 Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu Thr Val Gln 290 295 300 GAC TGC AAC TGC TCA ATC TAT CCC GGC CAC GTG TCA GGT CAC CGC ATG 1260 Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met 305 310 315 GCT TGG GAT ATG ATG ATG AAC TGG TCG CCT ACA ACA GCC CTG GTG GTA 1308 Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val 320 325 330 TCG CAG TTA CTC CGG ATC CCA CAA GCC ATC GTG GAC ATG GTG GCA GGG 1356 Ser Gln Leu Leu Arg Ile Pro Gln Ala Ile Val Asp Met Val Ala Gly 335 340 345 350 GCC CAC TGG GGA GTC CTG GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG 1404 Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly 355 360 365 AAC TGG GCT AAG GTC TTG ATT GTG ATG CTA CTC TTT GCT GGC GTT GAT 1452 Asn Trp Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp 370 375 380 GGG GGT ACC CAC GTG TCG GGG GGG AGC GCG GCC CAA ACC ACC AGC GGG 1500 Gly Gly Thr His Val Ser Gly Gly Ser Ala Ala Gln Thr Thr Ser Gly 385 390 395 CTT GCG TCC CTC TTT ACA TCC GGG TCG GCC CAG AAC ATC CAA CTT GTA 1548 Leu Ala Ser Leu Phe Thr Ser Gly Ser Ala Gln Asn Ile Gln Leu Val 400 405 410 AAC ACT AAC GGC AGC TGG CAC ATC AAC AGA ACT GCT CTG AAT TGC AAT 1596 Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn 415 420 425 430 GAC TCC CTC AAG ACT GGG TTC CTT GCC GCG CTG TTC TAC ACG CGC AAG 1644 Asp Ser Leu Lys Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr Arg Lys 435 440 445 TTC AAC TCG TCC GGA TGC CCA GAG CGC ATG GCC AGC TGC CGC CCC ATT 1692 Phe Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile 450 455 460 GAC AAG TTC GCT CAG GGG TGG GGT CCC ATT ACT CAT GTT GAG CCT CAC 1740 Asp Lys Phe Ala Gln Gly Trp Gly Pro Ile Thr His Val Glu Pro His 465 470 475 ATT TCA GAC CAG AGG CCT TAT TGC TGG CAC TAC GCG CCT CGG CCG TGC 1788 Ile Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys 480 485 490 GGT ATC GTA CCC GCG TCG CAG GTG TGT GGT CCA GTG TAT TGC TTC ACC 1836 Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr 495 500 505 510 CCG AGC CCT GTT GTG GTG GGA ACG ACC GAC CGC TTC GGT GTC CCC ACG 1884 Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr 515 520 525 TAC AAA TGG GGA GAG AAT GAG ACG GAC GTG CTA CTC CTC AAC AAC ACG 1932 Tyr Lys Trp Gly Glu Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr 530 535 540 CGG CCG CCG CAA GGC AAC TGG TTC AGC TGC ACA TGG ATG AAC AGC ACC 1980 Arg Pro Pro Gln Gly Asn Trp Phe Ser Cys Thr Trp Met Asn Ser Thr 545 550 555 GGG TTC ACC AGG ACG TGC GGG GGC CCC CCG TGT AAC ATC GGG GGG ACC 2028 Gly Phe Thr Arg Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Thr 560 565 570 GGC AAC GAC ACC TTG ACC TGC CCT ACG GAT TGC TTC CGT AAG CAC CCC 2076 Gly Asn Asp Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro 575 580 585 590 GAG GCC ACT TAC GCC AAA TGC GGC TCG GGG CCT TGG TTG ACA CCT AGG 2124 Glu Ala Thr Tyr Ala Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg 595 600 605 TGC TTA GTT GAC TAC CCA TAC AGA CTT TGG CAC TGC CCC TGC ACT GTC 2172 Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Cys Pro Cys Thr Val 610 615 620 AAT TTT ACC ATC TTC AAG GTC AGG ATG TAC GTG GGG GGT GTG GAG CAC 2220 Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His 625 630 635 AGG CTC GAC GCC GCG TGC AAT TGG ACT CGA GGA GAG CGC TGT GAT TTG 2268 Arg Leu Asp Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu 640 645 650 GAG GAC AGG GAT AGA TCA GAG CTC AGT CCG CTG CTA CTG TCT ACT ACA 2316 Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr 655 660 665 670 GAG TGG CAG ATA CTG CCT TGC TCC TTC ACC ACC CTA CCG GCT CTG TCC 2364 Glu Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser 675 680 685 ACC GGG TTG ATC CAC CTC CAT CAG AAC ATC GCG GAC GTG CAA TAC CTG 2412 Thr Gly Leu Ile His Leu His Gln Asn Ile Ala Asp Val Gln Tyr Leu 690 695 700 TAC GGT GTA GGG TCA GCG TTC GTC TCC GTC GTA GTC AGA TGG GAG TAC 2460 Tyr Gly Val Gly Ser Ala Phe Val Ser Val Val Val Arg Trp Glu Tyr 705 710 715 GTC CTG CTG CTC TTC CTT CTC CTG GCG GAC GCG CAT GTC TGC GCT TGC 2508 Val Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala His Val Cys Ala Cys 720 725 730 CTC TGG ATG ATG CTG CTG ATA GCC CAG GCT GAG GCC GCC TTA GAG AAC 2556 Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn 735 740 745 750 CTG GTG ATC CTC AAC GCG GCG TCC GTG GCT GGA GCG CAT GGC ATT CTC 2604 Leu Val Ile Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu 755 760 765 TCC TTC CTT GTG TTC TTC TGC GCT GCC TGG TAC ATC AAG GGC AAG CTG 2652 Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Lys Leu 770 775 780 GTG CCC GGG GCG GCA TAT GCT TTT TAT GGC GTA TGG CCG CTG CTC CTG 2700 Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu 785 790 795 CTC CTG CTG GCG TTA CCA CCA CGA GCA TAC GCC ATG GAC CGG GAG ATG 2748 Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met 800 805 810 GCC GCA TCG TGC GAA GGC GCG GTT TTC ATA GGT CTG GCA CTC TTG ACT 2796 Ala Ala Ser Cys Glu Gly Ala Val Phe Ile Gly Leu Ala Leu Leu Thr 815 820 825 830 TTG TCA CCA CAC TAC AAA GTG TTC CTC GCT AAG CTC ATA TGG TGG TTG 2844 Leu Ser Pro His Tyr Lys Val Phe Leu Ala Lys Leu Ile Trp Trp Leu 835 840 845 CAA TAT TTT ATC ACC AGG GCC GAG GCG TGC TTG CAA GTG TGG GTT CCC 2892 Gln Tyr Phe Ile Thr Arg Ala Glu Ala Cys Leu Gln Val Trp Val Pro 850 855 860 CCT CTC ATC GTT CGG GGG GGC CGC GAT GCC ATC ATC CTC CTC ACA TGC 2940 Pro Leu Ile Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Thr Cys 865 870 875 ATG GTC CAC CCA GAG CTA ATT TTT GAA ATC ACC AAA ATC TTG CTC GCC 2988 Met Val His Pro Glu Leu Ile Phe Glu Ile Thr Lys Ile Leu Leu Ala 880 885 890 ATA CTC GGT CCG CTC ATG GTG CTC CAG GCT GGC CTA ACT AGA GTG CCG 3036 Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Leu Thr Arg Val Pro 895 900 905 910 TAC TTC GTG CGC GCT CAA GGG CTC ATT CGT GTG TGC ATG TTG GTG CGG 3084 Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Val Cys Met Leu Val Arg 915 920 925 AAA GCC GCT GGG GGT CAT TAT GTC CAA ATG GCC CTC GTG AAG CTG GCC 3132 Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Leu Val Lys Leu Ala 930 935 940 GCA TTG ACG GGT ACG TAC GTA TAC AAC CAT CTT ACT CCA CTG CGA GAC 3180 Ala Leu Thr Gly Thr Tyr Val Tyr Asn His Leu Thr Pro Leu Arg Asp 945 950 955 TGG GCC CAT GCA GGC CTG CGC GAC CTT GTG GTG GCA GTT GAG CCT GTC 3228 Trp Ala His Ala Gly Leu Arg Asp Leu Val Val Ala Val Glu Pro Val 960 965 970 ATC TTC TCT GAC ATG GAG ACC AAG ATC ATC ACC TGG GGG GCA GAC ACC 3276 Ile Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr 975 980 985 990 GCA GCG TGC GGG GAC ATC ATC TCG GGT CTA CCC GTC TCC GCC CGA AGG 3324 Ala Ala Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg 995 1000 1005 GGG AGG GAG ATA CTT CTG GGA CCT GCC GAC AGC TTT AGG GAG CAG GGG 3372 Gly Arg Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Arg Glu Gln Gly 1010 1015 1020 TGG CGA CTC CTT GCG CCT ATC ACG GCC TAT TCC CAA CAG ACG CGG GGC 3420 Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly 1025 1030 1035 CTA ATT GGC TGC ATC ATC ACC AGC CTA ACT GTC CGG GAC AA 3461 Leu Ile Gly Cys Ile Ile Thr Ser Leu Thr Val Arg Asp 1040 1045 1050 配列番号：２配列の長さ：3401 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： ATCACTCCCC TGTGAGGAAC TACTGTCTTC ACGCAGAAAG CGTCTAGCCA TGGCGTTAGT 60 ATGAGTGTCG TGCAGCCTCC AGGACCCCCC CTCCCGGGAG AGCCATAGTG GTCTGCGGAA 120 CCGGTGAGTA CACCGGAATT GCCAGGACGA CCGGGTCCTT TCTTGGATCA ACCCGCTCAA 180 TGCCTGGAGA TTTGGGCGTG CCCCCGCGAG ACTGCTAGCC GAGTAGTGTT GGGTCGCGAA 240 AGGCCTTGTG GTACTGCCTG ATAGGGTGCT TGCGAGTGCC CCGGGAGGTC TCGTAGACCG 300 TGCATC ATG AGC ACA AAT CCC AAA CCC CAA AGA AAA ACC AAA CGT AAC 348 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn 1 5 10 ACC AAC CGT CGC CCA CAG GAC GTC AAG TTC CCG GGT GGT GGT CAG ATC 396 Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile 15 20 25 30 GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG 444 Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val 35 40 45 CGC GCG ACT AGG AAG ACT TCC GAG CGG TCA CAA CCT CGT GGA AGG CGA 492 Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg 50 55 60 CAA CCT ATC CCC AAG GCT CGC CAG CCC GAG GGC AGG GCC TGG GCT CAG 540 Gln Pro Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala Trp Ala Gln 65 70 75 CCC GGG TAC CCT TGG CCC CTC TAT GGC AAC GAG GGC ATG GGG TGG GCA 588 Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala 80 85 90 GGA TGG CTC CTG TCA CCC CGC GGC TCC CGG CCT AGT TGG GGC CCC ACG 636 Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr 95 100 105 110 GAC CCC CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC CTC 684 Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu 115 120 125 ACA TGC GGC TTC GCC GAC CTC ATG GGG TAC ATT CCG CTC GTC GGC GCC 732 Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala 130 135 140 CCC CTA GGG GGC GTT GCC AGG GCC CTG GCA CAT GGT GTC CGG GTT GTG 780 Pro Leu Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Val 145 150 155 GAG GAC GGC GTG AAC TAT GCA ACA GGG AAT TTG CCC GGT TGC TCT TTC 828 Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe 160 165 170 TCT ATC TTC CTC TTG GCT CTG CTG TCC TGT TTG ACC ATC CCA GCT TCC 876 Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser 175 180 185 190 GCT TAT GAG GTG CGC AAC GTA TCC GGG ATA TAC CAT GTC ACG AAC GAC 924 Ala Tyr Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp 195 200 205 TGC TCC AAC TCA AGT ATT GTG TAT GAG GCA GCG GAC ATG ATC ATG CAT 972 Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His 210 215 220 ACC CCC GGG TGC GTG CCC TGC GTT CGG GAG GGC AAC TCC TCC CGT TGC 1020 Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ser Ser Arg Cys 225 230 235 TGG GTG GCA CTT ACT CCC ACG CTA GCG GCC AGG AAT GCC AGC GTC CCC 1068 Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro 240 245 250 ACT ACG GCA ATA CGA CGC CAT GTC GAT TTG CTC GTT GGG GCG GCT GCT 1116 Thr Thr Ala Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala 255 260 265 270 TTC TGC TCC GCT ATG TAT GTG GGA GAT CTC TGC GGA TCT GTT TTC CTT 1164 Phe Cys Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu 275 280 285 GTC TCC CAG CTG TTC ACC TTC TCG CCC CGC CGG CAT GAG ACA ATA CAG 1212 Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Ile Gln 290 295 300 GAC TGC AAT TGC TCA ATC TAT CCC GGC CAC GTG TCA GGT CAC CGC ATG 1260 Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met 305 310 315 GCT TGG GAC ATG ATG ATG AAC TGG TCG CCT ACA ACG GCC CTG GTG GTG 1308 Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val 320 325 330 TCG CAG TTA CTC CGG ATC CCA CAA GCT ATC GTG GAC ATG GTG GCG GGG 1356 Ser Gln Leu Leu Arg Ile Pro Gln Ala Ile Val Asp Met Val Ala Gly 335 340 345 350 GCT CAC TGG GGT GTC CTA GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG 1404 Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly 355 360 365 AAC TGG GCT AAG GTA TTG ATT GTG ATG CTA CTT TTT GCC GGC GTC GAC 1452 Asn Trp Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp 370 375 380 GGG GAG ACC CGT GTG ACA GGG GGG CAG ATA GCC AGA AAT GCC TAC TCG 1500 Gly Glu Thr Arg Val Thr Gly Gly Gln Ile Ala Arg Asn Ala Tyr Ser 385 390 395 CTC ACG ACC CTC TTT TCA TCT GGG TCG GCT CAG AAC ATC CAG CTC ATA 1548 Leu Thr Thr Leu Phe Ser Ser Gly Ser Ala Gln Asn Ile Gln Leu Ile 400 405 410 AAC ACC AAC GGT AGC TGG CAC ATC AAC AGG ACT GCC CTG AAC TGC AAT 1596 Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn 415 420 425 430 GAC TCC CTC AAC ACC GGG TTT CTT GCC GCG CTG TTC TAC ACG CAC AAG 1644 Asp Ser Leu Asn Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Lys 435 440 445 TTC AAC GCG TCC GGA TGT CCA GAG CGC TTG GCC AGC TGC CGC CCC ATT 1692 Phe Asn Ala Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Pro Ile 450 455 460 GAC AAG TTC GAT CAG GGG TGG GGT CCC ATC ACT TAT GCT GAG CAG GGC 1740 Asp Lys Phe Asp Gln Gly Trp Gly Pro Ile Thr Tyr Ala Glu Gln Gly 465 470 475 GGC CAG GAC CAG AGG CCT TAT TGC TGG CAC TAC GCA CCT AAA CCA TGT 1788 Gly Gln Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Lys Pro Cys 480 485 490 GGT ATT GTA TCC GCG TCG AAG GTG TGT GGT CCA GTG TAT TGT TTC ACC 1836 Gly Ile Val Ser Ala Ser Lys Val Cys Gly Pro Val Tyr Cys Phe Thr 495 500 505 510 CCA AGC CCA GTT GTA GTG GGG ACG ACC GAT CGG TTC GGT GTC CCT ACG 1884 Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr 515 520 525 TAT AGC TGG GGG GAG AAT GAG ACA GAC GTG CTG CTC CTT AAC AAC ACG 1932 Tyr Ser Trp Gly Glu Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr 530 535 540 CGG CCG CCG CAA GGC AAC TGG TTC GGC TGT ACG TGG ATG AAC GGC ACT 1980 Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr 545 550 555 GGG TTC ACC AAG ACA TGC GGG GGC CCC CCG TGT AAC ATC GGG GGG GGC 2028 Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Gly 560 565 570 GGC AAT AAC ACC TTG ACC TGC CCT ACG GAC TGT TTC CGG AAG CAC CCC 2076 Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro 575 580 585 590 GCG GCC ACT TAC ACA AAA TGT GGT TCG GGA CCT TGG CTG ACA CCC AGG 2124 Ala Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg 595 600 605 TGC TTG GTA GAC TAC CCA TAC AGG CTC TGG CAC TAC CCC TGC ACT GCC 2172 Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ala 610 615 620 AAC TTT ACC ATC TTC AAG GTT AGG ATG TAT GTA GGG GGC GTG GAG CAC 2220 Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His 625 630 635 AGG CTC GAT GCT GCA TGC AAT TGG ACC CGA GGG GAA CGT TGC AAC TTG 2268 Arg Leu Asp Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu 640 645 650 GAG GAT AGG GAT AGA TTG GAG CTC AGC CCG CTA CTG CTG TCT ACA ACA 2316 Glu Asp Arg Asp Arg Leu Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr 655 660 665 670 GAG TGG CAG GTG CTG CCC TGT TCT TTC ACC ACC CTA CCG GCT CTG TCC 2364 Glu Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser 675 680 685 ACT GGT TTA ATT CAT CTC CAT CAG AAC ATC GTG GAC GTG CAA TAC CTG 2412 Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu 690 695 700 TAC GGT ATA GGG TCG GCA GTT GTT TCC TTT GCA ATC AAA TGG GAC TAT 2460 Tyr Gly Ile Gly Ser Ala Val Val Ser Phe Ala Ile Lys Trp Asp Tyr 705 710 715 ATC GTG ATA CTT TTC CTC CTC CTG GCG GAC GCG CGC GTC TGT GCC TGC 2508 Ile Val Ile Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys 720 725 730 TTG TGG ATG ATG CTG CTG ATA GCC CAG GCC GAG GCC GCC TTA GAA AAC 2556 Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn 735 740 745 750 CTG GTG GTC CTC AAT GCG GCG TCC GTG GCC GGA GCG CAT GGC ATT CTC 2604 Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu 755 760 765 TCC TTC CTT GTG TTC TTC TGT GCC GCC TGG TAC ATC AAG GGC AAG CTG 2652 Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Lys Leu 770 775 780 GTC CCC GGG GCA GCA TAT GCT TTC TAT GGA GTA TGG CCG CTG CTC CTG 2700 Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu 785 790 795 CTT CTG CTG GCC TTA CCA CCA CGA GCT TAC GCT ATG GAG CGG GAG ATG 2748 Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Glu Arg Glu Met 800 805 810 GCT GCA TCG TGC GGA GGC GCG GTG TTT GTA GGT CTG GTA CTC TTG ACT 2796 Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr 815 820 825 830 TTG TCA CCA TAC TAT AAA GAG TTC CTC GCC AGG CTC ATA TGG TGG TTG 2844 Leu Ser Pro Tyr Tyr Lys Glu Phe Leu Ala Arg Leu Ile Trp Trp Leu 835 840 845 CAA TAT TTT ATC ACC AGA GCC GAG GCG CAC CTG CAA GTG TGG ATC CCC 2892 Gln Tyr Phe Ile Thr Arg Ala Glu Ala His Leu Gln Val Trp Ile Pro 850 855 860 CCC CTC AAC ATT CGG GGG GGC CGC GAT GCC ATC ATC CTC CTC GCG TGT 2940 Pro Leu Asn Ile Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Ala Cys 865 870 875 GTA GTC CAC CCA GAG CTA ATC TTT GAC ATC ACC AAA CTC CTG CTC GCC 2988 Val Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Ala 880 885 890 ATA CTC GGT CCG CTC ATG GTG CTC CAG GCT AGC ATA ACT CAA GTG CCG 3036 Ile Leu Gly Pro Leu Met Val Leu Gln Ala Ser Ile Thr Gln Val Pro 895 900 905 910 TAC TTC GTA CGC GCC CAA GGG CTC ATT CGT GCA TGC ATG TTG GTG CGG 3084 Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Met Leu Val Arg 915 920 925 AAG GTT GCC GGG GGC CAT TAT GTC CAA ATG GCC TTT GTG AAG CTG ACC 3132 Lys Val Ala Gly Gly His Tyr Val Gln Met Ala Phe Val Lys Leu Thr 930 935 940 GCA CTG ACA GGT ACG TAC GTT TAT GAC CAT CTA ACT CCA CTG CGG GAC 3180 Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Arg Asp 945 950 955 TGG GCC CAC GCG GGC CTG CGA GAC CTC GCG GTG GCA GTA GAG CCC GTT 3228 Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val 960 965 970 GTC TTC TCT GAC ATG GAG ACC AAG GTC ATC ACC TGG GGG GCA GAC ACC 3276 Val Phe Ser Asp Met Glu Thr Lys Val Ile Thr Trp Gly Ala Asp Thr 975 980 985 990 GCG GCG TGT GGG GAC ATT ATC TTG GGT CTA CCT GTC TCC GCC CGA AGG 3324 Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg 995 1000 1005 GGT AGG GAG ATA CTT CTG GGG CCG GCC GAT AGT CTT GAA GGG CAG GGG 3372 Gly Arg Glu Ile Leu Leu Gly Pro Ala Asp Ser Leu Glu Gly Gln Gly 1010 1015 1020 TGG CGG CTC CTT GCG CCT ATC ACG GCC TA 3401 Trp Arg Leu Leu Ala Pro Ile Thr Ala 1025 １０３０ SEQ ID NO: 1 Array length: 3461 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: cDNA to genomic RNA Fragment type: N-terminal fragment Array: ATCACTCCCC TGTGAGGAAC TACTGTCTTC ACGCAGAAAG CGTCTAGCCA TGGCGTTAGT 60 ATGAGTGTCG TGCAGCCTCC AGGACCCCCC CTCCCGGGAG AGCCATAGTG GTCTGCGGAA 120 CCGGTGAGTA CACCGGAATT GCCAGGACGA CCGGGTCCTT TCTTGGATTA ACCCGCTCAA 180 TGCCTGGAGA TTTGGGCGTG CCCCCGCGAG ACTGCTAGCC GAGTAGTGTT GGGTCGCGAA 240 AGGCCTTGTG GTACTGCCTG ATAGGGTGCT TGCGAGTGCC CCGGGAGGTC TCGTAGACCG 300 TGCATC ATG AGC ACA AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC 348 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn 1 5 10 ACC AAC CGC CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT GGT CAG ATC 396 Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile 15 20 25 30 GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG 444 Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val 35 40 45 CGC GCG ACT AGG AAG ACT TCC GAG CGG TCG CAA CCT CGT GGA AGG CGA 492 Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg 50 55 60 CAA CCT ATC CCC AAG GCT CGC CGG CCC GAG GGC AGG GCC TGG GCT CAG 540 Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln 65 70 75 CCC GGG TAC CCT TGG CCC CTC TAT GGT AAC GAG GGC CTG GGG TGG GCA 588 Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala 80 85 90 GGA TGG CTC CTG TCA CCC CGC GGC TCC CGG CCT AGT TGG GGC CCC ACG 636 Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr 95 100 105 110 GAC CCC CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC CTC 684 Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu 115 120 125 ACA TGC GGC TTC GCC GAC CTC ATG GGG TAC ATT CCG CTC GTC GGC GCC 732 Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala 130 135 140 CCC CTA GGA GGC GTT GCC AGG GCC CTG GCG CAT GGC GTC CGG GTT CTG 780 Pro Leu Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu 145 150 155 GAA GAC GGC GTG AAT TAC GCA ACA GGG AAT CTG CCC GGT TGC TCT TTC 828 Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe 160 165 170 TCT ATC TTC CTC TTG GCT TTG CTG TCC TGT CTG ACC ATC CCA GCT TCC 876 Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser 175 180 185 190 GCT TAT GAA GTG CGC AAC GTG TCC GGG GTG TAC CAT GTC ACA AAC GAC 924 Ala Tyr Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp 195 200 205 TGC TCC AAC TCA AGT ATT GTG TAT GAG GCA GCG GAC GTG ATC ATG CAC 972 Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val Ile Met His 210 215 220 ACC CCC GGG TGC GTG CCC TGC GTT CGG GAG AGC AAT TTC TCC CGC TGC 1020 Thr Pro Gly Cys Val Pro Cys Val Arg Glu Ser Asn Phe Ser Arg Cys 225 230 235 TGG GTA GCG CTC ACT CCC ACG CTC GCG GCC AGA AAC AGC AGC ATC CCC 1068 Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ser Ser Ile Pro 240 245 250 ACT ACG ACA ATA CGA CGC CAT GTC GAT TTG CTC GTT GGG GCA GCT GCT 1116 Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala 255 260 265 270 CTC TGC TCC GCC ATG TAC GTG GGG GAT CTC TGC GGA TCT GTC TTC CTC 1164 Leu Cys Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu 275 280 285 GTC TCC CAG CTG TTC ACC TTC TCA CCT CGC CGG TAT GAG ACG GTA CAG 1212 Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu Thr Val Gln 290 295 300 GAC TGC AAC TGC TCA ATC TAT CCC GGC CAC GTG TCA GGT CAC CGC ATG 1260 Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met 305 310 315 GCT TGG GAT ATG ATG ATG AAC TGG TCG CCT ACA ACA GCC CTG GTG GTA 1308 Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val 320 325 330 TCG CAG TTA CTC CGG ATC CCA CAA GCC ATC GTG GAC ATG GTG GCA GGG 1356 Ser Gln Leu Leu Arg Ile Pro Gln Ala Ile Val Asp Met Val Ala Gly 335 340 345 350 GCC CAC TGG GGA GTC CTG GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG 1404 Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly 355 360 365 AAC TGG GCT AAG GTC TTG ATT GTG ATG CTA CTC TTT GCT GGC GTT GAT 1452 Asn Trp Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp 370 375 380 GGG GGT ACC CAC GTG TCG GGG GGG AGC GCG GCC CAA ACC ACC AGC GGG 1500 Gly Gly Thr His Val Ser Gly Gly Ser Ala Ala Gln Thr Thr Ser Gly 385 390 395 CTT GCG TCC CTC TTT ACA TCC GGG TCG GCC CAG AAC ATC CAA CTT GTA 1548 Leu Ala Ser Leu Phe Thr Ser Gly Ser Ala Gln Asn Ile Gln Leu Val 400 405 410 AAC ACT AAC GGC AGC TGG CAC ATC AAC AGA ACT GCT CTG AAT TGC AAT 1596 Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn 415 420 425 430 GAC TCC CTC AAG ACT GGG TTC CTT GCC GCG CTG TTC TAC ACG CGC AAG 1644 Asp Ser Leu Lys Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr Arg Lys 435 440 445 TTC AAC TCG TCC GGA TGC CCA GAG CGC ATG GCC AGC TGC CGC CCC ATT 1692 Phe Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile 450 455 460 GAC AAG TTC GCT CAG GGG TGG GGT CCC ATT ACT CAT GTT GAG CCT CAC 1740 Asp Lys Phe Ala Gln Gly Trp Gly Pro Ile Thr His Val Glu Pro His 465 470 475 ATT TCA GAC CAG AGG CCT TAT TGC TGG CAC TAC GCG CCT CGG CCG TGC 1788 Ile Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys 480 485 490 GGT ATC GTA CCC GCG TCG CAG GTG TGT GGT CCA GTG TAT TGC TTC ACC 1836 Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr 495 500 505 510 CCG AGC CCT GTT GTG GTG GGA ACG ACC GAC CGC TTC GGT GTC CCC ACG 1884 Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr 515 520 525 TAC AAA TGG GGA GAG AAT GAG ACG GAC GTG CTA CTC CTC AAC AAC ACG 1932 Tyr Lys Trp Gly Glu Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr 530 535 540 CGG CCG CCG CAA GGC AAC TGG TTC AGC TGC ACA TGG ATG AAC AGC ACC 1980 Arg Pro Pro Gln Gly Asn Trp Phe Ser Cys Thr Trp Met Asn Ser Thr 545 550 555 GGG TTC ACC AGG ACG TGC GGG GGC CCC CCG TGT AAC ATC GGG GGG ACC 2028 Gly Phe Thr Arg Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Thr 560 565 570 GGC AAC GAC ACC TTG ACC TGC CCT ACG GAT TGC TTC CGT AAG CAC CCC 2076 Gly Asn Asp Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro 575 580 585 590 GAG GCC ACT TAC GCC AAA TGC GGC TCG GGG CCT TGG TTG ACA CCT AGG 2124 Glu Ala Thr Tyr Ala Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg 595 600 605 TGC TTA GTT GAC TAC CCA TAC AGA CTT TGG CAC TGC CCC TGC ACT GTC 2172 Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Cys Pro Cys Thr Val 610 615 620 AAT TTT ACC ATC TTC AAG GTC AGG ATG TAC GTG GGG GGT GTG GAG CAC 2220 Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His 625 630 635 AGG CTC GAC GCC GCG TGC AAT TGG ACT CGA GGA GAG CGC TGT GAT TTG 2268 Arg Leu Asp Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu 640 645 650 GAG GAC AGG GAT AGA TCA GAG CTC AGT CCG CTG CTA CTG TCT ACT ACA 2316 Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr 655 660 665 670 GAG TGG CAG ATA CTG CCT TGC TCC TTC ACC ACC CTA CCG GCT CTG TCC 2364 Glu Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser 675 680 685 ACC GGG TTG ATC CAC CTC CAT CAG AAC ATC GCG GAC GTG CAA TAC CTG 2412 Thr Gly Leu Ile His Leu His Gln Asn Ile Ala Asp Val Gln Tyr Leu 690 695 700 TAC GGT GTA GGG TCA GCG TTC GTC TCC GTC GTA GTC AGA TGG GAG TAC 2460 Tyr Gly Val Gly Ser Ala Phe Val Ser Val Val Val Arg Trp Glu Tyr 705 710 715 GTC CTG CTG CTC TTC CTT CTC CTG GCG GAC GCG CAT GTC TGC GCT TGC 2508 Val Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala His Val Cys Ala Cys 720 725 730 CTC TGG ATG ATG CTG CTG ATA GCC CAG GCT GAG GCC GCC TTA GAG AAC 2556 Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn 735 740 745 750 CTG GTG ATC CTC AAC GCG GCG TCC GTG GCT GGA GCG CAT GGC ATT CTC 2604 Leu Val Ile Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu 755 760 765 TCC TTC CTT GTG TTC TTC TGC GCT GCC TGG TAC ATC AAG GGC AAG CTG 2652 Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Lys Leu 770 775 780 GTG CCC GGG GCG GCA TAT GCT TTT TAT GGC GTA TGG CCG CTG CTC CTG 2700 Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu 785 790 795 CTC CTG CTG GCG TTA CCA CCA CGA GCA TAC GCC ATG GAC CGG GAG ATG 2748 Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met 800 805 810 GCC GCA TCG TGC GAA GGC GCG GTT TTC ATA GGT CTG GCA CTC TTG ACT 2796 Ala Ala Ser Cys Glu Gly Ala Val Phe Ile Gly Leu Ala Leu Leu Thr 815 820 825 830 TTG TCA CCA CAC TAC AAA GTG TTC CTC GCT AAG CTC ATA TGG TGG TTG 2844 Leu Ser Pro His Tyr Lys Val Phe Leu Ala Lys Leu Ile Trp Trp Leu 835 840 845 CAA TAT TTT ATC ACC AGG GCC GAG GCG TGC TTG CAA GTG TGG GTT CCC 2892 Gln Tyr Phe Ile Thr Arg Ala Glu Ala Cys Leu Gln Val Trp Val Pro 850 855 860 CCT CTC ATC GTT CGG GGG GGC CGC GAT GCC ATC ATC CTC CTC ACA TGC 2940 Pro Leu Ile Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Thr Cys 865 870 875 ATG GTC CAC CCA GAG CTA ATT TTT GAA ATC ACC AAA ATC TTG CTC GCC 2988 Met Val His Pro Glu Leu Ile Phe Glu Ile Thr Lys Ile Leu Leu Ala 880 885 890 ATA CTC GGT CCG CTC ATG GTG CTC CAG GCT GGC CTA ACT AGA GTG CCG 3036 Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Leu Thr Arg Val Pro 895 900 905 910 TAC TTC GTG CGC GCT CAA GGG CTC ATT CGT GTG TGC ATG TTG GTG CGG 3084 Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Val Cys Met Leu Val Arg 915 920 925 AAA GCC GCT GGG GGT CAT TAT GTC CAA ATG GCC CTC GTG AAG CTG GCC 3132 Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Leu Val Lys Leu Ala 930 935 940 GCA TTG ACG GGT ACG TAC GTA TAC AAC CAT CTT ACT CCA CTG CGA GAC 3180 Ala Leu Thr Gly Thr Tyr Val Tyr Asn His Leu Thr Pro Leu Arg Asp 945 950 955 TGG GCC CAT GCA GGC CTG CGC GAC CTT GTG GTG GCA GTT GAG CCT GTC 3228 Trp Ala His Ala Gly Leu Arg Asp Leu Val Val Ala Val Glu Pro Val 960 965 970 ATC TTC TCT GAC ATG GAG ACC AAG ATC ATC ACC TGG GGG GCA GAC ACC 3276 Ile Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr 975 980 985 990 GCA GCG TGC GGG GAC ATC ATC TCG GGT CTA CCC GTC TCC GCC CGA AGG 3324 Ala Ala Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg 995 1000 1005 GGG AGG GAG ATA CTT CTG GGA CCT GCC GAC AGC TTT AGG GAG CAG GGG 3372 Gly Arg Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Arg Glu Gln Gly 1010 1015 1020 TGG CGA CTC CTT GCG CCT ATC ACG GCC TAT TCC CAA CAG ACG CGG GGC 3420 Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly 1025 1030 1035 CTA ATT GGC TGC ATC ATC ACC AGC CTA ACT GTC CGG GAC AA 3461 Leu Ile Gly Cys Ile Ile Thr Ser Leu Thr Val Arg Asp 1040 1045 1050 SEQ ID NO: 2 Array length: 3401 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Array: ATCACTCCCC TGTGAGGAAC TACTGTCTTC ACGCAGAAAG CGTCTAGCCA TGGCGTTAGT 60 ATGAGTGTCG TGCAGCCTCC AGGACCCCCC CTCCCGGGAG AGCCATAGTG GTCTGCGGAA 120 CCGGTGAGTA CACCGGAATT GCCAGGACGA CCGGGTCCTT TCTTGGATCA ACCCGCTCAA 180 TGCCTGGAGA TTTGGGCGTG CCCCCGCGAG ACTGCTAGCC GAGTAGTGTT GGGTCGCGAA 240 AGGCCTTGTG GTACTGCCTG ATAGGGTGCT TGCGAGTGCC CCGGGAGGTC TCGTAGACCG 300 TGCATC ATG AGC ACA AAT CCC AAA CCC CAA AGA AAA ACC AAA CGT AAC 348 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn 1 5 10 ACC AAC CGT CGC CCA CAG GAC GTC AAG TTC CCG GGT GGT GGT CAG ATC 396 Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile 15 20 25 30 GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG 444 Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val 35 40 45 CGC GCG ACT AGG AAG ACT TCC GAG CGG TCA CAA CCT CGT GGA AGG CGA 492 Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg 50 55 60 CAA CCT ATC CCC AAG GCT CGC CAG CCC GAG GGC AGG GCC TGG GCT CAG 540 Gln Pro Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala Trp Ala Gln 65 70 75 CCC GGG TAC CCT TGG CCC CTC TAT GGC AAC GAG GGC ATG GGG TGG GCA 588 Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala 80 85 90 GGA TGG CTC CTG TCA CCC CGC GGC TCC CGG CCT AGT TGG GGC CCC ACG 636 Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr 95 100 105 110 GAC CCC CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC CTC 684 Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu 115 120 125 ACA TGC GGC TTC GCC GAC CTC ATG GGG TAC ATT CCG CTC GTC GGC GCC 732 Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala 130 135 140 CCC CTA GGG GGC GTT GCC AGG GCC CTG GCA CAT GGT GTC CGG GTT GTG 780 Pro Leu Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Val 145 150 155 GAG GAC GGC GTG AAC TAT GCA ACA GGG AAT TTG CCC GGT TGC TCT TTC 828 Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe 160 165 170 TCT ATC TTC CTC TTG GCT CTG CTG TCC TGT TTG ACC ATC CCA GCT TCC 876 Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser 175 180 185 190 GCT TAT GAG GTG CGC AAC GTA TCC GGG ATA TAC CAT GTC ACG AAC GAC 924 Ala Tyr Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp 195 200 205 TGC TCC AAC TCA AGT ATT GTG TAT GAG GCA GCG GAC ATG ATC ATG CAT 972 Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His 210 215 220 ACC CCC GGG TGC GTG CCC TGC GTT CGG GAG GGC AAC TCC TCC CGT TGC 1020 Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ser Ser Arg Cys 225 230 235 TGG GTG GCA CTT ACT CCC ACG CTA GCG GCC AGG AAT GCC AGC GTC CCC 1068 Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro 240 245 250 ACT ACG GCA ATA CGA CGC CAT GTC GAT TTG CTC GTT GGG GCG GCT GCT 1116 Thr Thr Ala Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala 255 260 265 270 TTC TGC TCC GCT ATG TAT GTG GGA GAT CTC TGC GGA TCT GTT TTC CTT 1164 Phe Cys Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu 275 280 285 GTC TCC CAG CTG TTC ACC TTC TCG CCC CGC CGG CAT GAG ACA ATA CAG 1212 Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Ile Gln 290 295 300 GAC TGC AAT TGC TCA ATC TAT CCC GGC CAC GTG TCA GGT CAC CGC ATG 1260 Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met 305 310 315 GCT TGG GAC ATG ATG ATG AAC TGG TCG CCT ACA ACG GCC CTG GTG GTG 1308 Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val 320 325 330 TCG CAG TTA CTC CGG ATC CCA CAA GCT ATC GTG GAC ATG GTG GCG GGG 1356 Ser Gln Leu Leu Arg Ile Pro Gln Ala Ile Val Asp Met Val Ala Gly 335 340 345 350 GCT CAC TGG GGT GTC CTA GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG 1404 Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly 355 360 365 AAC TGG GCT AAG GTA TTG ATT GTG ATG CTA CTT TTT GCC GGC GTC GAC 1452 Asn Trp Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp 370 375 380 GGG GAG ACC CGT GTG ACA GGG GGG CAG ATA GCC AGA AAT GCC TAC TCG 1500 Gly Glu Thr Arg Val Thr Gly Gly Gln Ile Ala Arg Asn Ala Tyr Ser 385 390 395 CTC ACG ACC CTC TTT TCA TCT GGG TCG GCT CAG AAC ATC CAG CTC ATA 1548 Leu Thr Thr Leu Phe Ser Ser Gly Ser Ala Gln Asn Ile Gln Leu Ile 400 405 410 AAC ACC AAC GGT AGC TGG CAC ATC AAC AGG ACT GCC CTG AAC TGC AAT 1596 Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn 415 420 425 430 GAC TCC CTC AAC ACC GGG TTT CTT GCC GCG CTG TTC TAC ACG CAC AAG 1644 Asp Ser Leu Asn Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Lys 435 440 445 TTC AAC GCG TCC GGA TGT CCA GAG CGC TTG GCC AGC TGC CGC CCC ATT 1692 Phe Asn Ala Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Pro Ile 450 455 460 GAC AAG TTC GAT CAG GGG TGG GGT CCC ATC ACT TAT GCT GAG CAG GGC 1740 Asp Lys Phe Asp Gln Gly Trp Gly Pro Ile Thr Tyr Ala Glu Gln Gly 465 470 475 GGC CAG GAC CAG AGG CCT TAT TGC TGG CAC TAC GCA CCT AAA CCA TGT 1788 Gly Gln Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Lys Pro Cys 480 485 490 GGT ATT GTA TCC GCG TCG AAG GTG TGT GGT CCA GTG TAT TGT TTC ACC 1836 Gly Ile Val Ser Ala Ser Lys Val Cys Gly Pro Val Tyr Cys Phe Thr 495 500 505 510 CCA AGC CCA GTT GTA GTG GGG ACG ACC GAT CGG TTC GGT GTC CCT ACG 1884 Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr 515 520 525 TAT AGC TGG GGG GAG AAT GAG ACA GAC GTG CTG CTC CTT AAC AAC ACG 1932 Tyr Ser Trp Gly Glu Asn Glu Thr Asp Val Leu Leu Leu Asn Asn Thr 530 535 540 CGG CCG CCG CAA GGC AAC TGG TTC GGC TGT ACG TGG ATG AAC GGC ACT 1980 Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr 545 550 555 GGG TTC ACC AAG ACA TGC GGG GGC CCC CCG TGT AAC ATC GGG GGG GGC 2028 Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Gly 560 565 570 GGC AAT AAC ACC TTG ACC TGC CCT ACG GAC TGT TTC CGG AAG CAC CCC 2076 Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro 575 580 585 590 GCG GCC ACT TAC ACA AAA TGT GGT TCG GGA CCT TGG CTG ACA CCC AGG 2124 Ala Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg 595 600 605 TGC TTG GTA GAC TAC CCA TAC AGG CTC TGG CAC TAC CCC TGC ACT GCC 2172 Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ala 610 615 620 AAC TTT ACC ATC TTC AAG GTT AGG ATG TAT GTA GGG GGC GTG GAG CAC 2220 Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His 625 630 635 AGG CTC GAT GCT GCA TGC AAT TGG ACC CGA GGG GAA CGT TGC AAC TTG 2268 Arg Leu Asp Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu 640 645 650 GAG GAT AGG GAT AGA TTG GAG CTC AGC CCG CTA CTG CTG TCT ACA ACA 2316 Glu Asp Arg Asp Arg Leu Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr 655 660 665 670 GAG TGG CAG GTG CTG CCC TGT TCT TTC ACC ACC CTA CCG GCT CTG TCC 2364 Glu Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser 675 680 685 ACT GGT TTA ATT CAT CTC CAT CAG AAC ATC GTG GAC GTG CAA TAC CTG 2412 Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu 690 695 700 TAC GGT ATA GGG TCG GCA GTT GTT TCC TTT GCA ATC AAA TGG GAC TAT 2460 Tyr Gly Ile Gly Ser Ala Val Val Ser Phe Ala Ile Lys Trp Asp Tyr 705 710 715 ATC GTG ATA CTT TTC CTC CTC CTG GCG GAC GCG CGC GTC TGT GCC TGC 2508 Ile Val Ile Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys 720 725 730 TTG TGG ATG ATG CTG CTG ATA GCC CAG GCC GAG GCC GCC TTA GAA AAC 2556 Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn 735 740 745 750 CTG GTG GTC CTC AAT GCG GCG TCC GTG GCC GGA GCG CAT GGC ATT CTC 2604 Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu 755 760 765 TCC TTC CTT GTG TTC TTC TGT GCC GCC TGG TAC ATC AAG GGC AAG CTG 2652 Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Lys Leu 770 775 780 GTC CCC GGG GCA GCA TAT GCT TTC TAT GGA GTA TGG CCG CTG CTC CTG 2700 Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu 785 790 795 CTT CTG CTG GCC TTA CCA CCA CGA GCT TAC GCT ATG GAG CGG GAG ATG 2748 Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Glu Arg Glu Met 800 805 810 GCT GCA TCG TGC GGA GGC GCG GTG TTT GTA GGT CTG GTA CTC TTG ACT 2796 Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr 815 820 825 830 TTG TCA CCA TAC TAT AAA GAG TTC CTC GCC AGG CTC ATA TGG TGG TTG 2844 Leu Ser Pro Tyr Tyr Lys Glu Phe Leu Ala Arg Leu Ile Trp Trp Leu 835 840 845 CAA TAT TTT ATC ACC AGA GCC GAG GCG CAC CTG CAA GTG TGG ATC CCC 2892 Gln Tyr Phe Ile Thr Arg Ala Glu Ala His Leu Gln Val Trp Ile Pro 850 855 860 CCC CTC AAC ATT CGG GGG GGC CGC GAT GCC ATC ATC CTC CTC GCG TGT 2940 Pro Leu Asn Ile Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Ala Cys 865 870 875 GTA GTC CAC CCA GAG CTA ATC TTT GAC ATC ACC AAA CTC CTG CTC GCC 2988 Val Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Ala 880 885 890 ATA CTC GGT CCG CTC ATG GTG CTC CAG GCT AGC ATA ACT CAA GTG CCG 3036 Ile Leu Gly Pro Leu Met Val Leu Gln Ala Ser Ile Thr Gln Val Pro 895 900 905 910 TAC TTC GTA CGC GCC CAA GGG CTC ATT CGT GCA TGC ATG TTG GTG CGG 3084 Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Met Leu Val Arg 915 920 925 AAG GTT GCC GGG GGC CAT TAT GTC CAA ATG GCC TTT GTG AAG CTG ACC 3132 Lys Val Ala Gly Gly His Tyr Val Gln Met Ala Phe Val Lys Leu Thr 930 935 940 GCA CTG ACA GGT ACG TAC GTT TAT GAC CAT CTA ACT CCA CTG CGG GAC 3180 Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Arg Asp 945 950 955 TGG GCC CAC GCG GGC CTG CGA GAC CTC GCG GTG GCA GTA GAG CCC GTT 3228 Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val 960 965 970 GTC TTC TCT GAC ATG GAG ACC AAG GTC ATC ACC TGG GGG GCA GAC ACC 3276 Val Phe Ser Asp Met Glu Thr Lys Val Ile Thr Trp Gly Ala Asp Thr 975 980 985 990 GCG GCG TGT GGG GAC ATT ATC TTG GGT CTA CCT GTC TCC GCC CGA AGG 3324 Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg 995 1000 1005 GGT AGG GAG ATA CTT CTG GGG CCG GCC GAT AGT CTT GAA GGG CAG GGG 3372 Gly Arg Glu Ile Leu Leu Gly Pro Ala Asp Ser Leu Glu Gly Gln Gly 1010 1015 1020 TGG CGG CTC CTT GCG CCT ATC ACG GCC TA 3401 Trp Arg Leu Leu Ala Pro Ile Thr Ala 1025 1030

───────────────────────────────────────────────────── フロントページの続き (73)特許権者 000170565 国際試薬株式会社兵庫県神戸市西区高塚台４丁目３番２号 (74)上記３名の復代理人 100062007 弁理士川口義雄（外３名） (72)発明者八木慎太郎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 (72)発明者柏熊富子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 (72)発明者池口尚子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 (56)参考文献特開平４−253998（ＪＰ，Ａ) (58)調査した分野(Int.Cl.⁷，ＤＢ名) C07K 14/18 C12N 15/00 - 15/09 ＳｗｉｓｓＰｒｏｔ／ＰＩＲ／ＧｅｎｅＳｅｑＭＥＤＬＩＮＥ（ＳＴＮ)─────────────────────────────────────────────────── ─── Continuation of the front page (73) Patent holder 000170565 International Reagents Co., Ltd. 4-3-2 Takatsukadai, Nishi-ku, Kobe-shi, Hyogo (74) The above three sub-agents 100062007 Attorney Yoshio Kawaguchi (3 outside) (72) Inventor Shintaro Yagi 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Prefecture Tonen Co., Ltd. Research Institute (72) Tomiko Kashiwaguma 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Prefecture Tonen Research Institute Co., Ltd. (72) Inventor Naoko Ikeguchi 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Tonen Co., Ltd. Research Institute (56) Reference JP-A-4-253998 (JP, A) (58) ) Fields investigated (Int.Cl. ⁷ , DB name) C07K 14/18 C12N 15/00-15/09 SwissProt / PIR / GeneSeq MEDLINE (STN)

Claims

(57) [Claims]

1. A polypeptide consisting of the amino acid sequence from amino acid sequence SEQ ID NO: 384 NS1 antigen of HCV to 495.

2. A method for detecting a non-A non-B hepatitis virus antibody, which comprises reacting with the polypeptide according to claim 1 as an antigen.