JP2004000151A - Nucleic acid fragment encoding antigen of non-a non-b hepatitis virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid fragment containing a nucleotide sequence encoding an antigen of a structural or nonstructural region of a non-A non-B hepatitis virus obtained from plasma of a patient suffering from the non-A non-B hepatitis by genetic engineering technique, and to provide a method for producing the fragment. <P>SOLUTION: An RNA is extracted from the plasma of the patient suffering from the non-A non-B hepatitis, and a DNA is proliferated from the extract. The proliferated DNA is cloned by using a cloning vector which can replicate the DNA to determine the nucleotide sequence. As a result, 14 kinds of clones are obtained. The clones are useful for diagnosing a patient suffering from the non-A non-B hepatitis, and for detecting a carrier of the non-A non-B hepatitis virus. <P>COPYRIGHT: (C)2004,JPO

Description

【００２１】
【表２】

【００２２】
【表３】

【００２３】
【表４】

【００２４】
【表５】

【００２５】
【表６】

【００２６】
【表７】

【００２７】
【表８】

【００２８】
【表９】

【００２９】
【表１０】

【００３０】
【表１１】

【００３１】
【表１２】

【００３２】
【表１３】

【００３３】
【表１４】

【００３４】
これらの表より、クローンＣ１４−１は公表された非Ａ非Ｂ型肝炎ウイルス遺伝子との間で、ヌクレオチド配列で３．７〜１１．７％、アミノ酸配列で４．７〜１１．０％の相違を示した。またクローンＣ１４−２ではそれぞれ９．３〜３２．５％、９．２〜２７．１％；クローンＣ１４−３ではそれぞれ８．２〜３１．８％、２．５〜２４．５％；クローンＣ４−１ではそれぞれ９．６〜３３．４％、５．９〜２６．４％；クローンＣ４−２ではそれぞれ８．７〜２８．２％、３．９〜１４．８％；クローンＣ１４−４ではそれぞれ９．２〜３５．１％、７．６〜３４．３％；クローンＣ１４−５ではそれぞれ８．５〜２８．５％、４．７〜１３．９％；クローンＣ１４−６ではそれぞれ１２．２〜３６．５％、１３．８〜３７．２％；クローンＣ１４−７ではそれぞれ９．４〜３７．３％、４．９〜３３．４％；クローンＣ１４−８ではそれぞれ９．１〜３４．５％、３．２〜３０．２％；クローンＣ１４−９ではそれぞれ９．９〜３３．３％、４．３〜３１．２％；クローンＣ１４−１０ではそれぞれ１１．１〜４９．４％、９．２〜４１．２％；クローンＣ１４−１１ではそれぞれ６．４〜３０．３％、４．０〜２３．６％；クローンＣ１４−１２ではそれぞれ５．９〜３２．８％、４．７〜２７．４％の相違が認められた。このことは、Ｃ４、Ｃ１４株は現在までに公表されているＨＣＶ株とは別の株であることを示している。
【００３５】
従って、本発明は、非Ａ非Ｂ型肝炎患者血漿より遺伝子工学的手法により得られた非Ａ非Ｂ型肝炎ウイルスの構造及び非構造領域の抗原をコードするヌクレオチド配列を含む新規な核酸断片を提供する。
【００３６】
本発明の実施態様により、該核酸断片は、後記配列表中配列番号１，２，３，４，５，６，７，８，９，１０，１１，１２，１３および１４に示されるアミノ酸配列の全部または一部で表わされる非Ａ非Ｂ型肝炎ウイルス抗原をコードするヌクレオチド配列を含む。また、該ヌクレオチド配列には、遺伝暗号の縮重に基づく全ての配列が包含される。このようなヌクレオチド配列の具体例は、配列番号１に示されるヌクレオチド番号１から７００までの配列の全部または一部、配列番号２に示されるヌクレオチド番号１から９０９までの配列の全部または一部、配列番号３に示されるヌクレオチド番号１から８５２までの配列の全部または一部、配列番号４に示されるヌクレオチド番号１から８１９までの配列の全部または一部、配列番号５に示されるヌクレオチド番号３から９９２までの配列の全部または一部、配列番号６に示されるヌクレオチド番号１から５９４までの配列の全部または一部、配列番号７に示されるヌクレオチド番号２から１１４１までの配列の全部または一部、配列番号８に示されるヌクレオチド番号１から１１３４までの配列の全部または一部、配列番号９に示されるヌクレオチド番号２から１６６３までの配列の全部または一部、配列番号１０に示されるヌクレオチド番号２から６６７までの配列の全部または一部、配列番号１１に示されるヌクレオチド番号２から１１２０までの配列の全部または一部、配列番号１２に示されるヌクレオチド番号２から１１７４までの配列の全部または一部、配列番号１３に示されるヌクレオチド番号２から１０５７までの配列の全部または一部、配列番号１４に示されるヌクレオチド番号２から６４６までの配列の全部または一部である。
【００３７】
本発明はまた、上記核酸配列が、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された発現ベクターを提供する。さらに、本発明は該発現ベクターを含む宿主細胞を提供する。
【００３８】
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ーの他に、ウイルス（例えば、ワクシニアウイルス、バキュロウイルス等）が使用される。ＤＮＡ発現により得られる組換えペプチド又はポリペプチドが糖鎖構造をもつようにするか否かによって、使用し得るプロモーターおよび宿主の種類が決まる。すなわち、組換えペプチド又はポリペプチドが糖鎖構造を含まないようにする場合には、宿主として例えば大腸菌，枯草菌，放線菌等の原核生物を用いることができ、また、プロモーターとして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　，ＰＲ　等を用いることができる。この場合には、一般に他のペプチドとの融合体として得られるだろう。一方、組換えペプチド又はポリペプチドが糖鎖構造を含むようにする場合には、宿主として例えば酵母，植物細胞，昆虫細胞，動物細胞等の真核生物が挙げられ、またプロモーターとして酵母等に慣用のプロモーター例えば３−ホスホグリセレートキナーゼ，エノラーゼ等の解糖系酵素に対するプロモーターやアルコールデヒドロゲナーゼに対するプロモーター、哺乳動物細胞で使用され得るウイルスプロモーター例えばポリオーマウイルス，アデノウイルス，サルウイルスＳＶ４０，ワクシニアウイルス，サイトメガロウイルス等由来のプロモーターが挙げられる。
【００３９】
ベクターはさらに、形質転換された細胞の表現型選択を可能にするマーカー配列（例えばアンピシリン，テトラサイクリン耐性遺伝子等）、複製開始点、ターミネーター、リボソーム結合部位等を適宜含み得る。
【００４０】
本発明はさらに、組換え非Ａ非Ｂ型肝炎ウイルス抗原ペプチド又はポリペプチドの製造方法を提供する。この方法は、具体的には、本発明の上述の核酸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導入して形質転換体を得る工程、
前記核酸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ペプチド又はポリペプチドを発現させる工程、および
前記組換えペプチド又はポリペプチドを回収する工程
を包含する。
【００４１】
形質転換体の培養条件は、使用する宿主細胞に依存して決定され、増殖可能な培地、培養温度、培養時間等が適宜選択される。また、培養物からの組換えペプチド又はポリペプチドの精製は、慣用の技術例えば細胞の超音波破砕、可溶化抽出、硫安分画、各種クロマトグラフィー等により行うことができる。
【００４２】
本明細書中「組換えペプチド又はポリペプチド」とは、発現ベクターに組み込んだ非Ａ非Ｂ型肝炎ウイルス抗原をコードするＤＮＡを発現させて得られるペプチド又はポリペプチド自体または他のペプチド又はポリペプチドとの融合ペプチド又はポリペプチドを意味する。
【００４３】
本発明には、上記方法で得られた組換えペプチド又はポリペプチドも包含される。このようなペプチド又はポリペプチドは、慣用のペプチド合成技術を用いることによって化学合成することも可能であり、これは当業者には自明のことである。
【００４４】
本発明によって得られた組換えポリペプチドをＳＤＳ−ポリアクリルアミド電気泳動後、ウエスターンブロット法により正常人血清及び非Ａ非Ｂ型肝炎患者血清と反応させたところ、図４及び図５に示すように本組換えポリペプチドは非Ａ非Ｂ型肝炎患者血清とのみ反応した。従って本組換えポリペプチドは非Ａ非Ｂ型肝炎ウイルスに特異的な抗原であり、非Ａ非Ｂ型肝炎の診断及び非Ａ非Ｂ型肝炎ウイルスの検出に使用可能である。
【００４５】
【実施例】
以下の実施例により本発明をさらに詳細に説明するが、本発明はこれらの実施例に限定されない。
【００４６】
実施例１
ＲＴ−ＰＣＲによるＨＣＶ（＃Ｓ１４）遺伝子の検出
　慢性期非Ａ非Ｂ型肝炎患者の血漿よりＨＣＶ遺伝子をクローニングする方法として少量の血漿でクローニングが可能なＲＴ（リバース・トランスクリプターゼ）−ＰＣＲ法を利用してＨＣＶ遺伝子のクローニングを行った。
【００４７】
先ず、単一の慢性期の非Ａ非Ｂ型肝炎患者血漿（＃Ｓ１４）１００μｌに６ＭのＧＴＣ液（６Ｍグアニジンチオシアネート、３７．５ｍＭクエン酸ナトリウム、０．７５％ザルコシル、０．２Ｍメルカプトエタノール）２００μｌと酵母のｔ−ＲＮＡ（１０ｍｇ／ｍｌ）１μｌを加え撹拌する。更に３Ｍ酢酸ナトリウム（ｐＨ５．２）２０μｌ、ＴＥ飽和フェノール（ｐＨ７．５〜８．０）３０μｌ、クロロホルム／イソアミルアルコール（４９：１）７０μｌを加え素早く混合し、１０秒間撹拌した後、氷中に１５分間静置する。遠心機で１５０００　ｒｐｍ、２０分間４℃で遠心する。水層を採り、等量のイソプロピルアルコールと混合し−２０℃に１時間以上置く。これを１５０００　ｒｐｍ、２０分間４℃で遠心し、沈殿させる。沈殿物を４ＭのＧＴＣ（６Ｍ　ＧＴＣを滅菌水で希釈したもの）１００μｌに溶解し、等量のイソプロピルアルコールと混合し、−２０℃に１時間以上静置する。１５０００　ｒｐｍ、２０分間、４℃で遠心し沈殿物を得る。７０％エタノール１ｍｌで洗浄後、室温で風乾し、１０μｌの滅菌水に溶解しＲＮＡとして使用した。
【００４８】
ｃＤＮＡ合成はＲＮＡ１０μｌをシリコン処理チューブ（０．５ｍｌ）に分注した後、７０℃、３分間加熱し、氷上で急冷する。次にＲＮａｓｅインヒビター（宝酒造）１μｌ（５０単位／μｌ）、ｄＮＴＰ（各２０ｍＭ）１μｌ、１００ｍＭ　ＤＴＴ、５×ＲＴ　ｂｕｆｆｅｒ　（２５０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８．５）、３７５ｍＭ　ＫＣｌ、１５ｍＭ　ＭｇＣｌ２　）４μｌ、ランダムオリゴヘキサマープライマー（１００ｐｍｏｌ／μｌ）１μｌ、逆転写酵素（ＢＲＬ）（２００単位／μｌ）１μｌを加え、滅菌水で計２０μｌに合わせる。４２℃で２時間反応後、９４℃で５分間加熱し酵素を失活させた。このｃＤＮＡを用いてＰＣＲを行った。ＰＣＲは検出ＤＮＡの増幅感度と特異性を挙げる為に２ステップ法を用いた。即ち、先ず２種のプライマーで１回目のＰＣＲをかける（１ｓｔ　ｓｔｅｐ　ＰＣＲ）。次にそのＰＣＲ産物のＤＮＡ配列の内側に存在する２種のプライマーを用いて２回目のＰＣＲをかける（２ｎｄ　ｓｔｅｐ　ＰＣＲ）方法である。
【００４９】
Ｃ１４−１領域、Ｃ１４−２領域、Ｃ１４−３領域、Ｃ１４−４領域、Ｃ１４−５領域、Ｃ１４−６領域、Ｃ１４−７領域、Ｃ１４−８領域、Ｃ１４−９領域、Ｃ１４−１０領域、Ｃ１４−１１領域、Ｃ１４−１２領域の１２の領域についてプライマーを合成し、２ステップ法に使用した。以下に使用したＰＣＲプライマーを記述する。尚、それぞれの領域は既報の配列（Ｊ．　Ｖｉｒｏｌ．　６５，　１１０５−１１１３　（１９９１）；　Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ　８７，　９５２４−９５２８　（１９９０）；　Ｖｉｒｕｓ　Ｇｅｎｅｓ　５：３，　２４３−２５９　（１９９１）；　Ｊ．　Ｇｅｎｅｒａｌ　Ｖｉｒｏｌ．　７２，　２６９７−２７０４　（１９９１））を参考に設定した。又それぞれの増幅領域と既報の配列（ＨＣ−Ｊ６）との位置関係を図１及び図２に示す。
【００５０】
Ｃ１４−１領域については、１ｓｔ　ＰＣＲに際してはプライマー１４−１：５’−ＣＧＡＴＴＧＧＧＧＧＣＧＡ−３’及び１４−２：５’−ＴＴＧＣＡＡＡＡＴＴＡＡＣＣＣＣＧＴＣＣＴＣＣＡＧ−３’を使用し、２ｎｄ　ＰＣＲにはプライマー１４−１と１４−３：５’−ＣＡＴＧＡＧＧＴＣＧＧＣＧＡＡＧＣＣＧＣ−３’を用いた。
【００５１】
Ｃ１４−２領域については、１ｓｔ　ＰＣＲはプライマー１４−８：５’−ＣＡＣＣＡＡＴＧＧＣＡＧＴＴＧＧＣＡＣＡＴＣＡＡＣ−３’と１４−９：５’−ＧＧＡＣＴＡＣＣＣＧＡＣＣＣＴＴＧＡＴＧＴＡＣＣＡ−３’を使用し、２ｎｄ　ＰＣＲはプライマー１４−１０：５’−ＣＴＧＴＴＣＴＡＣＡＣＣＣＡＣＡＧＣＴＴＣＡＡＣ−３’と１４−１１：５’−ＧＣＧＴＧＣＡＡＧＡＣＧＡＣＣＡＡＣＴＴＣＴＣＴＡ−３’を用いた。
【００５２】
Ｃ１４−３領域については、１ｓｔ　ＰＣＲはプライマー１４−１２：５’−ＧＡＧＣＧＧＡＧＡＣＡＧＣＴＧＣＴＴＧＣＧＧＧＧＡ−３’と１４−１３：５’−ＡＴＡＧＧＴＧＧＡＧＴＡＣＧＴＧＡＴＧＧＧ−３’を使用し、２ｎｄ　ＰＣＲはプライマー１４−１４：５’−ＴＴＣＣＣＧＴＧＴＣＣＧＣＣＣＧＡ−３’と１４−１３を用いた。
【００５３】
Ｃ１４−４領域については、１ｓｔ　ＰＣＲはプライマー１４−４：５’−ＴＧＧＧＣＡＧＧＡＴＧＧＣＴＣＣＴＧＴＣ−３’と１４−５：５’−ＧＣＣＧＴＴＧＴＡＧＧＴＧＡＣＣＡＧＴＴＣ−３’を使用し、２ｎｄ　ＰＣＲはプライマー１４−６：５’−ＴＧＧＧＴＡＡＧＧＴＣＡＴＣＧＡＴＡＣＣ−３’と１４−５を用いた。
【００５４】
Ｃ１４−５領域については、１ｓｔ　ＰＣＲはプライマー１４−１５：５’−ＣＴＧＧＴＡＧＴＧＧＡＡＡＧＡＧＣＡＣＣＡＡＡＧＴ−３’と１４−１６：５’−ＴＧＣＡＴＧＣＡＣＧＴＧＧＣＧＡＴＧＴＡ−３’を使用し、２ｎｄ　ＰＣＲはプライマー１４−１７：５’−ＴＣＧＣＧＴＡＴＧＣＣＧＣＴＣＡＧＧＧＧＴＡＣＡＡ−３’と１４−１８：５’−ＧＴＣＡＧＧＧＴＡＡＣＣＴＣＧＴＴＧＧＴＡ−３’を用いた。
【００５５】
さらに、Ｃ１４−６，Ｃ１４−７，Ｃ１４−８，Ｃ１４−９，Ｃ１４−１０，Ｃ１４−１１及びＣ１４−１２領域については、下記に示すＰＣＲプライマーを用いた。
【００５６】
【表１５】

（ＮはＧ，Ａ，Ｔ，Ｃのミックスを示す）。
【００５７】
ＰＣＲの条件は、０．５ｍｌチューブ中に上記ｃＤＮＡ合成反応液を２０μｌと１０×ＰＣＲ緩衝液（１００ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３）、５００ｍＭ　ＫＣｌ、１５ｍＭ　ＭｇＣｌ２　、０．１％　ｇｅｌａｔｉｎｅ　）８μｌ、１ｓｔ　ｓｔｅｐプライマー２種（各７５ｐｍｏｌｅ　）、２ｍＭ　ｄＮＴＰ　８μｌを加え、滅菌水で１００μｌにする。９４℃で１０分間加熱し、Ａｍｐｌｉ　Ｔａｑ　（Ｐｅｒｋｉｎ−Ｅｌｍｅｒ−Ｃｅｔｕｓ）１μｌ（５単位）を加え撹拌後、ミネラルオイルを重層し軽く遠心する。ＰＣＲ反応は、変性９４℃１分間、アニーリング５５℃１分間、伸長７２℃２分間の条件で３０サイクル行った。次に新しい０．５ｍｌチューブに１ｓｔ　ＰＣＲ反応終了液１０μｌ、１０×ＰＣＲ緩衝液９μｌを加え、２ｎｄ　ｓｔｅｐプライマー２種（各７５ｐ　ｍｏｌｅ）、２ｍＭ　ｄＮＴＰ９μｌ、滅菌水で１００μｌとする。９４℃で１０分間加熱し、Ａｍｐｌｉ　Ｔａｑ　１μｌ（５単位）を加え撹拌後、ミネラルオイルを重層し軽く遠心し、先の条件で２ｎｄ　ＰＣＲを行う。反応後、反応液１０μｌをアガロースゲル電気泳動し、特異的に増幅されたＤＮＡ断片を検出した。
ＰＣＲ産物（ＨＣＶ＃Ｓ１４のＤＮＡ断片）のクローニングと塩基配列の決定
　ＨＣＶ遺伝子は複製時に変異が導入され易い可能性が考えられた。そこでクローニング時に発生する人為的な変異をできるだけ少なくする為にベクターとしてｐＢＲ３２２（Ｓｕｔｃｈｌｉｆｆｅ，　Ｊ．　Ｇ．，　Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｓｙｍｐｏｓｉｕｍ，　４３，　７７−９０（１９７９））を改変したベクター（ｐＢＭ）を用いた。ｐＢＭはｐＢＲ３２２の制限酵素ＥｃｏＲ　ＶサイトからＢａｌ　Ｉサイトの間の配列を制限酵素で欠失させ、ＥｃｏＲ　ＩサイトとＨｉｎｄ　ＩＩＩサイト間にｐＵＣ１１９（Ｖｉｅｒｉａ，　Ｊ．，　Ｍｅｓｓｉｎｇ，　Ｊ．，　Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，　１５３，　３−１１　（１９８７））のマルチクローニングサイトのＥｃｏＲ　ＩサイトからＨｉｎｄ　ＩＩＩサイトまでを組み込んだ（ΔｐＢＲ　ＭＣＳ）。次にｐＢＲ３２２のＶｓｐ　ＩサイトからＳｃａ　Ｉサイトの間の配列をｐＵＣ１１９のＶｓｐＩサイトからＳｃａ　Ｉサイト間の配列に置き換え、この間のＰｓｔ　Ｉサイトを欠失させ全長３１２２ｂｐのベクターを作製した（図３）。
【００５８】
ＨＣＶのＤＮＡが検出されたＰＣＲ反応液は全量を等量のクロロホルム／イソアミルアルコール（２４：１）と混和し、遠心後、その水層を０．５ｍｌチューブに移し、１０分の１量の３Ｍ酢酸ナトリウム（ｐＨ５．２）、２倍量のエタノールを加え、エタノール沈殿した。沈殿物は１０ｍＭトリス塩酸−１ｍＭ　ＥＤＴＡ（ｐＨ７．４）（ＴＥ）３００μｌに溶解し、ウルトラフリーＣ３ＴＫ（日本ミリポアリミテッド）にて遠心濾過し、残存プライマー除去、および脱塩を行った。処理液は１０×Ｔ４　ＤＮＡポリメラーゼ緩衝液（３０ｍＭトリス酢酸、０．６６Ｍ酢酸カリウム、０．１Ｍ酢酸マグネシウム、５ｍＭ　ＤＴＴ、１ｍｇ／ｍｌ　ＢＳＡ）２μｌ、２ｍＭ　ｄＮＰＴ１μｌ、Ｔ４ＤＮＡポリメラーゼ４単位（宝酒造）を加え滅菌水にて２０μｌとし、１２℃、１５分間反応した。反応後、等量のフェノール／クロロホルム（２５：２４）、クロロホルム／イソアミルアルコール（２４：１）でそれぞれ１回ずつ抽出を行い、水層をエタノール沈殿した。沈殿物は、７５％エタノールで洗浄後、風乾し、１０×イミダゾール緩衝液（０．５Ｍイミダゾール塩酸（ｐＨ６．４）、０．１８Ｍ塩化マグネシウム、５０ｍＭ　ＤＴＴ）４μｌ、２４％ポリエチレングリコール６０００　１０μｌ、１０ｍＭ　ＡＴＰ　０．５μｌ、Ｔ４ＤＮＡキナーゼ（宝酒造）２０単位を加え滅菌水で４０μｌとし、３７℃、１時間反応して５’末端をリン酸化した。クロロホルム／イソアミルアルコール処理によって酵素を失活させ、水層をエタノール沈殿後、７５％エタノールで洗浄した。沈殿物は低融点アガロースゲル電気泳動によりＤＮＡを単離し、ＴＥ飽和フェノールで２回抽出を行い、ＤＮＡ断片をエタノール沈殿し７５％エタノールで洗浄後、滅菌水１０μｌに溶解しその１μｌをアガロースゲル電気泳動し、ＤＮＡ断片量を決定した。
【００５９】
ここで得られたＤＮＡ断片はあらかじめ制限酵素Ｓｍａ　Ｉにて切断し、アルカリフォスファターゼ処理によりその５’末端の脱リン酸化を行ったｐＢＭベクターとの連結反応を行う。
【００６０】
ｐＢＭ（２０μｌ）は制限酵素反応液５０μｌ（１０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）、７ｍＭ　ＭｇＣｌ２　、２０ｍＭ　ＫＣｌ、Ｓｍａ　Ｉ（宝酒造）８０単位）中で３０℃、９０分反応し、６８℃、１５分加熱後エタノール沈殿する。沈殿物を７５％エタノールで洗浄後、風乾し、１０×アルカリフォスファターゼ緩衝液（１００ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３）、１ｍＭ　ＺｎＣｌ２　、１０ｍＭ　ＭｇＣｌ２　）５μｌ、アルカリフォスファターゼ（牛小腸由来；宝酒造）１単位に滅菌水を加え５０μｌとし、３７℃、１時間反応させることにより脱リン酸化した。５００ｍＭ　ＥＤＴＡ（ｐＨ７．５）０．５μｌ、１０％ＳＤＳ２．５μｌを加え、更にプロテアーゼＫを終濃度５０μｇ／ｍｌとなるように加え、５６℃、３０分反応し、酵素を失活させた後、低融点アガロースゲル電気泳動によりベクターを単離し、ＴＥ飽和フェノールで２回抽出を行い、エタノール沈殿して、７５％エタノールで洗浄、風乾後、滅菌水５０μｌに溶解した。その１μｌをアガロースゲル電気泳動し、ベクター量を決定し、終濃度０．１μｇ／ｍｌのＳｍａ　Ｉクローニングベクターとした。
【００６１】
リン酸化したＤＮＡ断片はＳｍａ　Ｉクローニングベクター２５ｎｇに対してモル比で１５倍から２０倍量加え、１０×ライゲーション緩衝液（０．６６Ｍ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）、５０ｍＭ　ＭｇＣｌ２　、５０ｍＭ　ＤＴＴ）２μｌ、１０ｍＭヘキサミン塩化コバルト２μｌ、ＢＳＡ（１ｍｇ／ｍｌ）２μｌ、１０ｍＭ　ＡＴＰ１μｌ、Ｔ４ＤＮＡリガーゼ（宝酒造）３５０単位を加え、滅菌水で２０μｌとし、１６℃で一夜連結反応を行った。この反応液にｔＲＮＡ（１０ｍｇ／ｍｌ）０．５μｌを加え、エタノール沈殿後、７５％エタノールで洗浄し、その沈殿物を１０μｌの滅菌水に溶解し、その半量を用いて大腸菌ＪＭ１０９株、又はＳＣＳ１株を形質転換した。形質転換に用いる感受性大腸菌株（コンピテントセル）は既報（Ｊ．　Ｍｏｌ．　Ｂｉｏｌ．，　１６６，　５７７　（１９８３））に基づいて調製したものを用いた。
【００６２】
形質転換菌はＬＢ−Ａｍｐプレート（１％バクトトリプトン、０．５％酵母エキス、０．５％塩化ナトリウム、１．５％寒天、アンピシリン５０μｇ／ｍｌ）上で一夜培養した後、プレート上に出現したコロニーをそれぞれ３ｍｌ　ＬＢ−Ａｍｐの入った１５ｍｌチューブで培養し、１．５ｍｌの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（Ｍａｎｉａｔｉｓら，　Ｍｏｌｅｃｕｌｅｒ　Ｃｌｏｎｉｎｇ　：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，　１９８２　）を行い、１５μｌのＤＮＡ液を調製した。内、２〜３μｌを制限酵素ＥｃｏＲ　ＩとＨｉｎｄ　ＩＩＩ　各４単位、反応緩衝液（５０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、１０ｍＭ　ＭｇＣｌ２　、１ｍＭ　ＤＴＴ、１００ｍＭ　ＮａＣｌ）１０μｌ中で３７℃、１時間反応させた後、アガロースゲル電気泳動を行い、挿入されたＤＮＡ断片の大きさを確認した。
【００６３】
各１２の領域はＣ１４−１が約７１０ｂｐ、Ｃ１４−２領域が約９５０ｂｐ、Ｃ１４−３領域が約８５０ｂｐ、Ｃ１４−４領域が約６００ｂｐ、Ｃ１４−５領域が約１２００ｂｐ、Ｃ１４−６領域が約１１３４ｂｐ、Ｃ１４−７領域が約１６６４ｂｐ、Ｃ１４−８領域が約６６７ｂｐ、Ｃ１４−９領域が約１１２０ｂｐ、Ｃ１４−１０領域が約１１７４ｂｐ、Ｃ１４−１１領域が約１０５７ｂｐ、Ｃ１４−１２領域が約６４８ｂｐのＤＮＡ断片がそれぞれ確認された。
【００６４】
得られた１２種類のＤＮＡは更にＳａｎｇｅｒ等のジデオキシターミネーション法（Ｓｃｉｅｎｃｅ，　２１４，　１２０５−１２１０　（１９８１））を用い、その塩基配列を決定した。又、このＤＮＡ塩基配列決定に使用したそれぞれの領域クローンをＣ１４−１、Ｃ１４−２、Ｃ１４−３、Ｃ１４−４、Ｃ１４−５、Ｃ１４−６、Ｃ１４−７、Ｃ１４−８、Ｃ１４−９、Ｃ１４−１０、Ｃ１４−１１、Ｃ１４−１２と命名した。又、決定した遺伝子の塩基配列及びそれより推定されるアミノ酸配列をＣ１４−１は配列番号１として、Ｃ１４−２は配列番号２、Ｃ１４−３は配列番号３、Ｃ１４−４は配列番号６、Ｃ１４−５は配列番号７、Ｃ１４−６は配列番号８、Ｃ１４−７は配列番号９、Ｃ１４−８は配列番号１０、Ｃ１４−９は配列番号１１、Ｃ１４−１０は配列番号１２、Ｃ１４−１１は配列番号１３、Ｃ１４−１２は配列番号１４として示した。上記プラスミドは形質転換体としてＣ１４−１は微工研菌寄第１３０２９　号、Ｃ１４−２は同第１３０３０　号、Ｃ１４−３は同第１３０３１　号、Ｃ１４−４は同第１３０３２　号、Ｃ１４−５は同第１３０３３　号として平成４年６月２４日付けで、また、Ｃ１４−６はＦＥＲＭ　Ｐ−１３５９２　、Ｃ１４−８は同Ｐ−１３５９３　、Ｃ１４−９は同Ｐ−１３５９４　、Ｃ１４−１０は同Ｐ−１３５９５　、Ｃ１４−１１は同Ｐ−１３５９６　、Ｃ１４−１２は同Ｐ−１３５９７　として平成５年４月９日付けで工業技術院生命工学工業技術研究所に寄託されている。
【００６５】
実施例２
ＲＴ−ＰＣＲによるＨＣＶ（＃４）遺伝子の検出
　上記実施例１で示したと同様の方法にて、実施例１とは異なる単一の慢性非Ａ非Ｂ型肝炎患者血漿からのＨＣＶ（＃４）遺伝子のＲＴ−ＰＣＲを行い、Ｃ４−１及びＣ４−２領域の増幅ＤＮＡ断片を検出した。
【００６６】
用いたプライマーを以下に示した。
【００６７】
Ｃ４−１領域については、１ｓｔ　ＰＣＲはプライマー４−１：５’−ＡＴＧＧＡＧＡＣＴＡＡＡＣＴＣＡＴＣＡＣ−３’と４−２：５’−ＡＣＴＧＴＧＣＣＧＡＴＧＣＣＣＡＡＧＡＴ−３’を使用し、２ｎｄ　ＰＣＲはプライマー４−３：５’−ＴＡＣＴＴＣＴＡＧＧＡＣＣＧＧＣＣＧＡＴ−３’と１４−１３：５’−ＡＴＡＧＧＴＧＧＡＧＴＡＣＧＴＧＡＴＧＧＧ−３’を用いた。
【００６８】
Ｃ４−２領域については、１ｓｔ　ＰＣＲはプライマー４−４：５’−ＴＧＧＡＧＣＧＴＡＴＡＴＧＴＣＣＡＡＧＧ−３’と４−５：５’−ＧＡＣＡＴＧＣＡＴＧＣＣＡＴＧＡＴＧＴＡ−３’を使用し、２ｎｄ　ＰＣＲはプライマー４−４と４−６：５’−ＣＡＣＡＴＴＴＧＧＴＣＣＣＡＣＧＡＴＧＧ−３’を用いた。
ＰＣＲ産物のクローニングと塩基配列の決定
　クローニングに際しては、ベクターとしてｐＵＣ１１９を用い、そのＳｍａ　Ｉサイトと、上記プライマーを用いてＰＣＲにより増幅した遺伝子断片を実施例１で示した方法によってクローニングし、塩基配列を決定した。又、このＤＮＡ塩基配列決定に使用したそれぞれの領域クローンをＣ４−１、Ｃ４−２と命名した。決定した遺伝子の塩基配列及びそれより推定されるアミノ酸配列をＣ４−１は配列番号４として、Ｃ４−２は配列番号５として示した。上記プラスミドは形質転換体としてＣ４−１は微工研菌寄第１３０２７　号、Ｃ４−２は同第１３０２８　号として平成４年６月２４日付で寄託されている。
【００６９】
実施例３
大腸菌を用いたＨＣＶ（＃Ｓ１４）由来遺伝子の発現（その１）
ａ）発現プラスミドの構築
上記実施例１において得られたクローンＣ１４−１のＤＮＡを利用してプライマーＢ１：５’−ＣＡＴＧＡＧＣＡＴＡＡＡＴＣＣＴＡＡＡＣＣＴＣＡＡＡＧ−３’とＢ２：５’−ＡＴＣＴＧＣＡＧＴＴＡＴＡＧＧＧＴＧＴＣＧＡＴＧＡＣＣＴＴＡＣＣＣ−３’を用いて実施例１のＰＣＲ条件でＰＣＲを行い、約３８０ｂｐのＤＮＡ断片を増幅した。ＰＣＲ反応液は全量を実施例１の方法でクロロホルム／イソアミルアルコール（２４：１）で処理し、その水層をエタノール沈殿し、ＴＥ３００μｌに溶解後、遠心瀘過し、残存プライマー除去及び脱塩を行い、Ｔ４ＤＮＡポリメラーゼ処理後、Ｔ４ＤＮＡキナーゼ処理によって５’末端を燐酸化した。得られたＤＮＡ断片は反応緩衝液（５０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、１０ｍＭ　ＭｇＣｌ２　、１ｍＭ　ＤＴＴ、１００ｍＭ　ＮａＣｌ）中でＰｓｔ　Ｉ２０単位を加えて消化し、低融点アガロースゲル電気泳動を行った。アガロースゲルよりＤＮＡを単離し、ＴＥ飽和フェノールで２回抽出後、エタノール沈殿し、減菌水１０μｌに溶解し、約３８０ｂｐのＤＮＡ断片として精製した。ここで得られたＤＮＡ断片は発現ベクターｐＫＫ２２３−３（ファルマシア）をあらかじめ上述の条件にて制限酵素Ｐｓｔ　Ｉで切断し、更に実施例１に示した条件にて制限酵素Ｓｍａ　Ｉで切断し、アルカリフォスファターゼ処理によりその５’末端の脱燐酸化を行ったそのベクター２５ｎｇと実施例１の条件でＴ４ＤＮＡリガーゼにより連結反応を行い、大腸菌ＪＭ１０５株を用い、形質転換した。
【００７０】
形質転換菌はＬＢ−Ａｍｐプレート（１％バクトトリプトン、０．５％酵母エキス、０．５％ＮａＣｌ、１．５％寒天、５０μｇ／ｍｌアンピシリン）上で一夜培養後、プレート上に出現したコロニーをそれぞれ３ｍｌのＬＢ−Ａｍｐの入った１５ｍｌチューブで培養し、その１．５ｍｌ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（　Ｍａｎｉａｔｉｓ　ｅｔ　ａｌ，　Ｍｏｌｅｃｕｌｅｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，１９８２）　を行い、１５μｌのＤＮＡ液を調製した。内２−３μｌを制限酵素ＥｃｏＲ　ＩとＰｓｔ　Ｉ各４単位、反応緩衝液（５０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、１０ｍＭ　ＭｇＣｌ２　、ｌｍＭ　ＤＴＴ、１００ｍＭ　ＮａＣｌ）１０μｌ中で３７℃、１時間反応させた後、アガロースゲル電気泳動を行い、約３８０ｂｐのＤＮＡ断片が挿入されているクローンを得た。
ｂ）ウエスタンブロット法による発現の確認及び非Ａ非Ｂ型肝炎患者血清との反応
　上記大腸菌クローンを３ｍｌのＬＢ−Ａｍｐ培地で３７℃、３時間前培養した後、その５０μｌを新しいＬＢ−Ａｍｐ培地５ｍｌに接種し、３７℃、２時間培養した。培養液に終濃度２ｍＭになるようにＩＰＴＧ（和光純薬）を加え、更に３７℃、３時間培養した。培養液１．５ｍｌを１３０００ｒｐｍ　、２分間遠心し、集菌後、ＴＥ１ｍｌで菌を洗浄し、１３０００　ｒｐｍ、２分間遠心し、再び集菌した。集菌したペレットに５０μｌの滅菌水および５０μｌの２×サンプル緩衝液（１００ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８）、２０％グリセロール、１０％ＳＤＳ、５％　２−メルカプトエタノール、０．２％ブロムフェノルブルー）を加え懸濁混和し、懸濁液を１００℃、５分間煮沸後氷冷下で超音波処理し、−７０℃で凍結融解を２回繰り返しサンプルとした。
【００７１】
上記サンプル３０μｌをＭＩＮＩ　ＰＲＯＴＥＡＮ　ＩＩ　Ｄｕａｌ　ＳｌａｂＣｅｌｌ（Ｂｉｏｒａｄ）を用いてＬａｅｍｍｌｉの方法（Ｎａｔｕｒｅ，　２２７，　６８０（１９７０））に準じて１５ｍＡ，１．５時間ＳＤＳ−ポリアクリルアミドゲル電気泳動を行った。泳動後、ゲルを取り出し、ＰＶＤＦメンブレン（ミリポア）を密着させＭＩＮＩ　ＴＲＡＮＳ　ＢＬＯＴ　Ｅｌｅｃｔｒｏｐｈｏｒｅｔｉｃ　Ｔｒａｎｓｆｅｒ　Ｃｅｌｌ（Ｂｉｏｒａｄ）を用いて２５０ｍＡ、１．７５時間転写した。転写後、メンブレンを５％スキムミルク、２％ＢＳＡを含む緩衝液Ｉ’（１０ｍＭ　Ｎａ−ｐｈｏｓｐｈａｔｅ（ｐＨ７．０）、１％ＢＳＡ、０．１５Ｍ　ＮａＣｌ、２．５ｍＭ　ＥＤＴＡ）に浸漬し、室温で２時間ブロッキングした。５％スキムミルク、２％ＢＳＡを含む緩衝液Ｉ’で４０倍希釈した血清検体にブロッキングしたメンブレンを入れ、室温で４時間反応させた。反応後、メンブレンを緩衝液ＩＩ（１０ｍＭ　Ｎａ−ｐｈｏｓｐｈａｔｅ（ｐＨ７．０）、０．１５Ｍ　ＮａＣｌ、０．０５％Ｔｗｅｅｎ２０）で３回洗浄後、２％スキムミルクを含む緩衝液Ｉ’で１００ｍｕ／ｍｌに希釈した抗人ＩｇＧ−ＰＯＤ標識抗体液（ヤギ抗体）にいれ、室温で３０分間反応させた。反応後、メンブレンを取り出し、緩衝液ＩＩで５回洗浄した。洗浄したメンブレンを発色液（２０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、０．５Ｍ　ＮａＣｌ、０．０５％４−クロロ１ナフトール、０．０１８％Ｈ２　Ｏ２　、１６．７％メタノール）に浸漬し、室温で１５分間反応させた。
【００７２】
結果を図４に示した。図４においては、非Ａ非Ｂ型慢性肝炎患者血清５例（Ｎｏ．１〜Ｎｏ．　５）　と健常人血清５例（Ｎｏ．　６〜Ｎｏ．　１０）についてウエスタンブロットを行った結果を示したが、非Ａ非Ｂ型肝炎患者血清でのみ、全てに強い陽性反応が検出され、発現した抗原が、非Ａ非Ｂ型肝炎患者の診断及び非Ａ非Ｂ型肝炎ウイルスキャリヤーの検出に有用であることが示された。
【００７３】
実施例４
大腸菌を用いたＨＣＶ（＃Ｓ１４）由来遺伝子の発現（その２）
ａ）発現プラスミドの構築
実施例１において得られたクローンＣ１４−３とＣ１４−５のＤＮＡを緩衝液（Ｔａｋａｒａ　Ｕｎｉｖｅｒｓａｌ　ｂｕｆｆｅｒ　Ｈ　）中でそれぞれ制限酵素ＥｃｏＲＩ，ＰｓｔＩ及びＥｃｏＲＩで消化し、アガロースゲル電気泳動によりそれぞれ約９３０ｂｐと約９５０ｂｐのＤＮＡ断片を単離し、ＴＥ飽和フェノール及びクロロホルム処理後、エタノール沈殿し、滅菌水２５μｌに溶解することによって精製した。精製した各ＤＮＡをそれぞれ上記緩衝液中で制限酵素ＳｃａＩで消化し、アガロースゲル電気泳動によりそれぞれ約７８０ｂｐと９２０ｂｐのＤＮＡを単離し、ＴＥ飽和フェノール及びクロロホルム処理後、エタノール沈殿することによって精製した。精製した２種類のＤＮＡ、及び予め上記緩衝液中、制限酵素ＥｃｏＲＩで消化したベクターｐＢｌｕｅｓｃｒｉｐｔを混合し、Ｔ４　ＤＮＡリガーゼにより連結反応を行い、大腸菌ＪＭ１０９株を用い、形質転換した。
【００７４】
形質転換菌はＬＢ−Ａｍｐプレート（１％バクトトリプトン、０．５％酵母エキス、１％ＮａＣｌ、１．５％寒天、５０μｇ／ｍｌアンピシリン）上で一晩培養後、プレート上に出現したコロニーをそれぞれ３ｍｌのＬＢ−Ａｍｐの入った１５ｍｌチューブで培養し、その１．５ｍｌを遠心処理により集菌し、プラスミドのミニプレパレーション（Ｍａｎｉａｔｉｓ　ｅｔ　ａｌ，　Ｍｏｌｅｃｕｌｅｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，１９８２）を行い、２０μｌのＤＮＡ液を調製した。調製したＤＮＡ液の４μｌを緩衝液（Ｔａｋａｒａ　Ｕｎｉｖｅｒｓａｌ　Ｂｕｆｆｅｒ　Ｈ　）中ＥｃｏＲＩで消化し、アガロースゲル電気泳動することによって約１７００ｂｐのＤＮＡ断片が挿入されているクローン（２８−１４Ｄ）を得た。このクローン２８−１４ＤのＤＮＡを利用してプライマーＦ２：５’−ＣＡＧＡＡＴＴＣＡＴＧＧＡＡＡＣＡＣＴＣＧＡＣＡＴＣＧＣＣ−３’とプライマーＲ：５’−ＣＡＣＴＧＣＡＧＴＴＡＴＧＡＧＡＣＡＧＣＧＴＣＴＴＧＡＧＧＧＡＣ−３’を用いてＰＣＲ反応を行った。ＰＣＲ反応は、上記ＤＮＡ１μｌに１０×ＰＣＲ緩衝液（１００ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３）、５００ｍＭ　ＫＣｌ、１５ｍＭ　ＭｇＣｌ２　、０．１％ｇｅｒａｔｉｎｅ）５μｌ、プライマーＦ２、Ｒ（各２４０ｐＭ）、２５ｍＭ　ｄＮＴＰ　０．２μｌ、Ｔａｑ　ｐｏｌｙｍｅｒａｓｅ（Ｂｏｅｈｒｉｎｇｅｒ）０．２μｌ（１単位）を加え、滅菌水で５０μｌとした後撹拌し、ミネラルオイルを重層し、変性９４℃０．５分間、アニーリング５５℃０．５分間、伸長７２℃１分間の条件で４４サイクル行った。反応後、反応液の全量をＴＥ飽和フェノール及びクロロホルム処理し、その水層をエタノール沈殿し、滅菌水４０μｌに溶解し、緩衝液（Ｔａｋａｒａ　Ｕｎｉｖｅｒｓａｌ　Ｂｕｆｆｅｒ　Ｈ　）５μｌ、制限酵素ＥｃｏＲＩ２０単位、ＰｓｔＩ２０単位を加えて消化した。消化後１．５％アガロースゲル電気泳動により約８６０ｂｐのＤＮＡ断片を単離し、ＴＥ飽和フェノール及びクロロホルム処理後エタノール沈殿し、５μｌの滅菌水に溶解することにより精製ＤＮＡを得た。ここで得られたＤＮＡ断片はその１μｌを、発現ベクターｐＫＫ２２３−３（ファルマシア）を予め制限酵素ＥｃｏＲＩとＰｓｔＩで切断し、上述の条件で精製した物１μｌと混合し、Ｔ４ＤＮＡリガーゼにより連結し、大腸菌ＪＭ１０９株を用い、形質転換した。
【００７５】
形質転換菌は、ＬＢ−Ａｍｐプレート（１％バクトトリプトン、０．５％酵母エキス、１％ＮａＣｌ、１．５％寒天、５０μｇ／ｍｌアンピシリン）上で一晩培養後、プレート上に出現したコロニーをそれぞれ３ｍｌのＬＢ−Ａｍｐの入った１５ｍｌチューブで培養し、その１．５ｍｌを遠心処理により集菌し、プラスミドのミニプレパレーション（Ｍａｎｉａｔｉｓ　ｅｔ　ａｌ，　Ｍｏｌｅｃｕｌｅｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，１９８２）を行い、２０μｌのＤＮＡ液を調製した。調製したＤＮＡ液の４μｌを緩衝液（Ｔａｋａｒａ　Ｕｎｉｖｅｒｓａｌ　Ｂｕｆｆｅｒ　Ｈ　）中ＥｃｏＲＩ及びＰｓｔＩで消化し、アガロースゲル電気泳動することによって約８６０ｂｐのＤＮＡ断片が挿入されているクローンを得た。
ｂ）ウエスタンブロット法による発現の確認及び非Ａ非Ｂ型肝炎患者血清との反応
上記大腸菌クローンを３ｍｌのＬＢ−Ａｍｐ培地で３７℃、３時間前培養した後、その５０μｌを新しいＬＢ−Ａｍｐ培地５ｍｌに接種し、３７℃、２時間培養した。培養液に終濃度２ｍＭになるようにＩＰＴＧ（和光純薬）を加え、更に３７℃、３時間培養した。培養液１．５ｍｌを１３０００ｒｐｍ　、２分間遠心し集菌後、ＴＥ１ｍｌで菌を洗浄し、１３０００　ｒｐｍ、２分間遠心し、再び集菌した。集菌したペレットに５０μｌの滅菌水及び５０μｌの２×サンプル緩衝液（１００ｍＭＴｒｉｓ−ＨＣｌ（ｐＨ６．８）、２０％グリセロール、１０％ＳＤＳ、５％２−メルカプトエタノール、０．２％ブロムフェノルブルー）を加え懸濁混和し、懸濁液を１００℃、５分間煮沸後氷冷下で超音波処理し、−７０℃で凍結融解を２回繰り返しサンプルとした。
【００７６】
上記サンプルをＭＩＮＩ　ＰＲＯＴＥＡＮ　ＩＩ　Ｄｕａｌ　Ｓｌａｂ　Ｃｅｌｌ（Ｂｉｏｒａｄ）を用いてＬａｅｍｍｌｉの方法（Ｎａｔｕｒｅ，　２２７，　６８０（１９７０））に準じて１５ｍＡ，１．５時間ＳＤＳ−ポリアクリルアミドゲル電気泳動を行った。泳動後、ゲルを切り出し、ＰＶＤＦメンブレン（ミリポア）を密着させＭＩＮＩ　ＴＲＡＮＳ　ＢＬＯＴ　Ｅｌｅｃｔｒｏｐｈｏｒｅｔｉｃ　Ｔｒａｎｓｆｅｒ　Ｃｅｌｌ（Ｂｉｏｒａｄ）を用いて２５０ｍＡ、１．７５時間転写した。転写後、メンブレンを５％スキムミルク、２％ＢＳＡを含む緩衝液Ｉ’（１０ｍＭ　Ｎａ−ｐｈｏｓｐｈａｔｅ（ｐＨ７．０）、１％ＢＳＡ、０．１５Ｍ　ＮａＣｌ、２．５ｍＭ　ＥＤＴＡ）に浸漬し、室温で２時間ブロッキングした。５％スキムミルク、２％ＢＳＡを含む緩衝液Ｉ’で４０倍希釈した血清検体にブロッキングしたメンブレンを入れ、室温で４時間反応させた。反応後、メンブレンを緩衝液ＩＩ（１０ｍＭ　Ｎａ−ｐｈｏｓｐｈａｔｅ（ｐＨ７．０）、０．１５Ｍ　ＮａＣｌ、０．０５％Ｔｗｅｅｎ２０）で３回洗浄後、２％スキムミルクを含む緩衝液Ｉ’で１００ｍｕ／ｍｌに希釈した抗人ＩｇＧ−ＰＯＤ標識抗体液（ヤギ抗体）に入れ、室温で３０分間反応させた。反応後、メンブレンを取り出し、緩衝液ＩＩで５回洗浄した。洗浄したメンブレンを発色液（２０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、０．５Ｍ　ＮａＣｌ、０．０５％４−クロロ１ナフトール、０．０１８％Ｈ２　Ｏ２　、１６．７％メタノール）に浸漬し、室温で１５分間反応させた。
【００７７】
結果を図５に示した。図５においては、非Ａ非Ｂ型慢性肝炎患者血清５例（Ｎｏ．１〜Ｎｏ．　５）　と健常人血清５例（Ｎｏ．　６〜Ｎｏ．　１０）についてウエスタンブロットを行った結果を示したが、非Ａ非Ｂ型肝炎患者血清でのみ、全てに強い陽性反応が検出され、発現した抗原が、非Ａ非Ｂ型肝炎患者の診断に有用であることが示された。
【００７８】
【発明の効果】
本発明により、従来既報の配列と塩基配列、アミノ酸配列においてその相同性を異にするＨＣＶ遺伝子がクローニングされた。本発明によって得られた塩基配列は非Ａ非Ｂ型肝炎患者の核酸診断への利用が期待でき、又塩基配列をもとに作製されたポリペプチドは、非Ａ非Ｂ型肝炎患者の抗体測定に応用できる他、モノクローナル抗体を作製することにより抗原検出系への応用が期待できる。特に、エンベロープ領域においてはワクチンへの応用も考えられ、ＨＣＶ感染症の診断、予防に大きく貢献するものと考えられる。
【００７９】
【配列表】

【図面の簡単な説明】
【図１】この図は、ＰＣＲによるＨＣＶ＃Ｓ１４株および＃４株のクローニング戦略を示す。図中、各領域に付した上側の数字はＨＣ−Ｊ６上のヌクレオチド配列の位置を示し、また下側の数字は使用したＰＣＲプライマーの名称を示す。なお、括弧内の数字はＤＮＡ断片の長さ（ｂｐ）を示す。
【図２】この図は、ＰＣＲによるＨＣＶ＃Ｓ１４株のクローニング戦略を示す。図中、各領域に付した上側の数字はＨＣ−Ｊ６上のヌクレオチド配列の位置を示し、また下側の数字は使用したＰＣＲプライマーの名称を示す。なお、括弧内の数字はＤＮＡ断片の長さ（ｂｐ）を示す。
【図３】この図は、クローニング用ベクターｐＢＭの構築工程を示す。
【図４】この図は、慢性非Ａ非Ｂ型肝炎患者血清５例（Ｎｏ．１〜Ｎｏ．５）と健常人血清５例（Ｎｏ．６〜Ｎｏ．１０　）について本発明発現抗原（実施例３参照）を用いてウエスタンブロッティングを行った結果を示す写真である。
【図５】この図は、慢性非Ａ非Ｂ型肝炎患者血清５例（Ｎｏ．１〜Ｎｏ．５）と健常人血清５例（Ｎｏ．６〜Ｎｏ．１０）について本発明発現抗原（実施例４参照）を用いてウエスタンブロッティングを行った結果を示す写真である。レーンＭは分子量マーカーを示す。[0001]
[Industrial applications]
The present invention relates to nucleic acid fragments encoding antigens of the structural and nonstructural regions of non-A non-B hepatitis virus. The present invention also relates to an expression vector containing the nucleic acid fragment and a host cell containing the vector. The present invention further relates to a method for producing the antigenic peptide or polypeptide and a recombinant peptide or polypeptide obtained thereby.
[0002]
[Prior art]
In general, hepatitis A virus, which is mainly transmitted orally, and hepatitis B virus, which is transmitted via blood, are widely known as causative viruses of viral hepatitis. It is also known that there is a D-type (δ) hepatitis virus that accompanies infection with the hepatitis B virus.
[0003]
Apart from these, there was hepatitis for which infectivity was pointed out for a long time, but the presence of the pathogenic factor was unknown, and the existence of multiple viruses was suggested, but the presence of hepatitis A and B viruses and other Hepatitis was ruled out as a non-A non-B hepatitis by the exclusion diagnosis. Recently, a non-A non-B hepatitis virus that causes hepatitis by oral infection has been isolated and identified.
[0004]
On the other hand, non-A, non-B hepatitis, which is transmitted mainly through blood transfusion, can be prevented more than 90% of post-transfusion hepatitis by the development of vaccine and screening of blood for transfusion. In addition, more than 50% of infected people became chronic, and the rate of transition to liver cirrhosis and liver cancer was high, which was a serious problem.
[0005]
In 1989, Choo et al. Of the United States Chiron Co., USA cloned a viral gene by immunoscreening using chimpanzee plasma infected with human plasma as a sample, and expressed using a microorganism based on the cloned viral gene. A diagnostic method using an antibody test using an antigen has been developed (Science {244: 359-362} (1989); Science {244: 362-364} (1989); Japanese Translation of PCT International Publication No. 2-500880). With this as an opportunity, active research has begun around the world, and the entire primary structure of the virus has been elucidated (Proc. Natl. Acad. Sci. 87: 9524 (1990); Proc. Natl. Acad. Sci. 88: 2451 (1991); J. Virol. 65, 1105 (1991)), which is now widely referred to as Hepatitis C （virus (HCV). However, the reagent using the c100-3 antigen, which was initially developed by Chiron, could only detect 70-80% of patients with chronic non-A, non-B hepatitis (Shiro Iino et al., Medicine and Pharmacy # 26 (1): 87). -95, $ 1991). However, since the HCV gene has been actively cloned in the United States and Japan as well, the development of a second-generation reagent containing a core antigen, a virus structural protein, has enabled the detection of nearly 90% of patients. (Tada Kawai et al., Clinical Testing Equipment and Reagents # 14 (4): 725-733, $ 1991). However, even with this improved second generation reagent, only about 40% is detected in sporadic non-A non-B hepatitis patients.
[0006]
On the other hand, with the progress of HCV research, it has been pointed out that there is a significant difference in homology between viral genes, and it is becoming possible to divide them into at least two or more genotypes (Virus Gene 5: 3, @ 243 (1991); \ Proc. \ Natl. \ Acad. \ Sci. \ 88: 10292 (1991)).
[0007]
[Problems to be solved by the invention]
As noted above, a significant portion of non-A non-B hepatitis patients has been made diagnostic by second generation HCV antibody detection reagents that combine antigens in both structural and non-structural regions, but these reagents still provide diagnostics. Some patients cannot be detected. It is not clear whether this is due to a different type of non-A non-B hepatitis virus or to a completely different etiological agent.
[0008]
In addition, with the advent of treatment methods for non-A, non-B hepatitis patients such as interferon administration, not only detection of antibodies but also more significant measurement of genes and antigens for judgment of therapeutic effects has been required. It is strongly desired. However, it is becoming clear that non-A non-B hepatitis viruses have different types of groups, and it is also revealed that they have considerable diversity particularly in the putative envelope region. When performing antibody measurement, antigen measurement, and gene measurement as indicators of viral infection, it is considered necessary to consider the diversity of virus antigens and genes. It is considered necessary to obtain the expression product.
[0009]
It is an object of the present invention to provide novel nucleic acid fragments encoding antigens of the non-A non-B hepatitis virus structural and non-structural regions.
[0010]
Another object of the present invention is to provide an expression vector containing the nucleic acid fragment.
[0011]
Yet another object of the present invention is to provide a host cell containing the expression vector.
[0012]
Another object of the present invention is to provide a method for producing the antigenic peptide or polypeptide obtained by culturing the host cell and expressing the nucleic acid fragment.
[0013]
[Means for Solving the Problems]
The present inventors have succeeded in cloning a non-A non-B hepatitis virus gene different from the previously reported one from a specific non-A non-B hepatitis patient plasma in order to achieve the above object. Was completed.
[0014]
In completing the present invention, RNA is extracted from plasma of a non-A non-B hepatitis patient, and cDNA is obtained by the action of reverse transcriptase to obtain cDNA using two kinds of primers (PCR (polymerase chain reaction; Science # 230: 1350 ( 1985)) to amplify the DNA. Primers used for amplification are described in the published sequences (J. Virol. $ 65, $ 1105 (1991); Proc. Natl. Acad. Sci. 87: 9524 (1990); Virus Gene 5: 3 243 (1991); General {Virol, {72: 2697 (1991)}). The amplified DNA was cloned using a cloning vector capable of replicating in Escherichia coli, and the nucleotide sequence was determined using Sanger's dideoxy chain termination method (Science, # 214, # 1205 (1981)).
[0015]
Fourteen types of clones were obtained by the above method, and C14-1, C14-2, C14-3, C4-1, C4-2, C14-4, C14-5, C14-6, C14-7, and C14-8, respectively. , C14-9, C14-10, C14-11 and C14-12. C14 and C4 are a series of clones obtained from a single patient, respectively. Of the 14 clones obtained, 13 clones other than the C14-7 clone were transferred to Escherichia coli JM109 strain, and then used as transformants as microtransformers No. 13029, No. 13030, and No. 13031. Nos. 13027, 13028, 13032, and 13033１３０ (deposited on June 24, 1992), and FERM P-13592, P-13593, and P It has been deposited with the Institute of Biotechnology and Industrial Technology of the National Institute of Advanced Industrial Science and Technology as-13594, P-13595, P-13596, and P-13597 (both deposited on April 9, 1993).
[0016]
As shown in FIG. 1 and FIG. 2, the obtained 14 types of clones showed that C14-1 was a 5 ′ untranslated region and a core region, respectively, by homology comparison with the nucleotide sequence of a previously reported non-A non-B hepatitis virus gene. C14-3, C4-1, C4-2 and C14-5 are in the NS3 region, C14-2 is in the E2 / NS1 region, C14-4 is the core / E1 region, and C14-6 is the core / E1 / E2 // The NS1 region, C14-7 were estimated to be NS2 / NS3 regions, C14-8 was estimated to be NS4 / NS3 regions, C14-9 was estimated to be NS4 / NS5 regions, and C14-10, C14-11 and C14-12 were estimated to be NS5 regions. The determined clones C14-1, C14-2, C14-3, C4-1, C4-2, C14-4, C14-5, C14-6, C14-7, C14-8, C14-9, C14-10 , C14-11 and C14-12 are shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 in the Sequence Listing below. It was shown to.
[0017]
The characteristics of each clone obtained are shown below.
(1) Clone @ C14-1
Consisting of 701 nucleotides, the translated region has nucleotide numbers from 320 to 700 (127 amino acids) and corresponds to the 5 'untranslated region and part of the core antigen region.
(2) Clone @ C14-2
Consisting of 910 nucleotides, the translation region has nucleotide numbers from 1 to 909 (303 amino acids) and corresponds to a part of the E2 / NS1 region.
(3) Clone @ C14-3
Consisting of 852 nucleotides, the translation region is 1-852 (284 amino acids) and corresponds to a part of the NS2 and NS3 antigen regions.
(4) Clone @ C4-1
The translation region consists of 819 nucleotides, and has a nucleotide number of 1 to 819 (273 amino acids), which corresponds to a part of the NS2 and NS3 antigen regions.
(5) Clone @ C4-2
Consisting of 992 nucleotides, the translation region is nucleotides 3 to 992 (330 amino acids) and corresponds to a part of the NS3 antigen region.
(6) Clone @ C14-4
The translation region consists of 596 nucleotides and has a nucleotide number of 1 to 594 (198 amino acids), which corresponds to the core antigen region and a part of the E1 antigen region.
(7) Clone @ C14-5
Consisting of 1143 nucleotides, the translation region is nucleotide numbers 2 to 1141 (380 amino acids) and corresponds to a part of the NS3 antigen region.
(8) Clone C14-6
Consisting of 1134 nucleotides, the translated region is nucleotides 1-1134 (378 amino acids) and corresponds to a portion of the E1 and core, E2 / NS1 regions.
(9) Clone @ C14-7
It consists of 1664 nucleotides, and the translation region is nucleotide numbers 2 to 1663 (554 amino acids) and corresponds to a part of the E2 / NS1, NS2, and NS3 regions.
(10) Clone @ C14-8
Consisting of 667 nucleotides, the translation region has nucleotide numbers from 2 to 667 (222 amino acids) and corresponds to a part of the NS4 and NS3 regions.
(11) Clone @ C14-9
Consisting of 1120 nucleotides, the translation region has nucleotide numbers 2-1120 (373 amino acids) and corresponds to a part of the NS4 and NS5 regions.
(12) Clone @ C14-10
It consists of 1174 nucleotides, and the translation region is nucleotide numbers 2 to 1174 (391 amino acids) and corresponds to a part of the NS5 region.
(13) Clone @ C14-11
The translation region consists of 1057 nucleotides, and has a nucleotide number of 2 to 1057 (352 amino acids), which corresponds to a part of the NS5 region.
(14) Clone @ C14-12
Consisting of 648 nucleotides, the translation region has nucleotide numbers from 2 to 646 (215 amino acids) and corresponds to a part of the NS5 region.
[0018]
The coding region of the non-A non-B hepatitis virus genome is composed of a core / envelope structural region and a non-structural region (NS), and CORE, E1, E2 / NS1, NS2 from the 5 ′ end of the coding region. , NS3, NS4, and NS5 in this order (J. Virology (1991), 65: 1105-1113).
[0019]
Furthermore, clones C14-1, C14-2, C14-3, C4-1, C4-2, C14-4, C14-5, C14-6, C14-7, C14-8, C14-9, C14-10, The nucleotide sequence and deduced amino acid sequence of C14-11 and C14-12 were respectively reported by HCV1 (Proc. Natl. Acad. Sci. (1991), {88: 2451-2455) and HCVBK (J. Virology (1991), 65: HCV-J1 (Proc. Natl. Acad. Sci. (1990), 87: 9524-9528), HC-J6 (J. General Virology (1991), 72: 2697-2704) and HC-J8 (1105). Virology @ (1992), 188: 331-341 The results of the sequence comparison and homology) shown in the table below 1,2,3,4,5,6,7,8,9,10,11,12,13 and 14.
[0020]
[Table 1]

[0021]
[Table 2]

[0022]
[Table 3]

[0023]
[Table 4]

[0024]
[Table 5]

[0025]
[Table 6]

[0026]
[Table 7]

[0027]
[Table 8]

[0028]
[Table 9]

[0029]
[Table 10]

[0030]
[Table 11]

[0031]
[Table 12]

[0032]
[Table 13]

[0033]
[Table 14]

[0034]
From these tables, clone C14-1 has a nucleotide sequence of 3.7-11.7% and an amino acid sequence of 4.7-11.0% of the published non-A non-B hepatitis virus gene. The differences are shown. 9.3-32.5% and 9.2-27.1% for clone C14-2; 8.2-31.8% and 2.5-24.5% for clone C14-3; clone 9.6-33.4%, 5.9-26.4% for C4-1 respectively; 8.7-28.2%, 3.9-14.8% for clone C4-2, respectively; Clone C14- 9.2 to 35.1% and 7.6 to 34.3%, respectively; 8.5 to 28.5%, 4.7 to 13.9% for clone C14-5; 12.3-36.5%, 13.8-37.2%, respectively; 9.4-37.3%, 4.9-33.4%, respectively, for clone C14-7; 9 for clone C14-8, respectively. 0.1-34.5%, 3.2-30.2%; 9 for clone C14-9, respectively. 9-33.3%, 4.3-31.2%; 11.1-49.4% and 9.2-41.2%, respectively, for clone C14-10; 6.4-, respectively, for clone C14-11. 30.3%, 4.0 to 23.6%; clone C14-12 showed a difference of 5.9 to 32.8% and 4.7 to 27.4%, respectively. This indicates that the C4 and C14 strains are different from the HCV strains published so far.
[0035]
Accordingly, the present invention provides a novel nucleic acid fragment containing a nucleotide sequence encoding a non-A non-B hepatitis virus structural and non-structural region antigen obtained from non-A non-B hepatitis patient plasma by genetic engineering techniques. provide.
[0036]
According to an embodiment of the present invention, the nucleic acid fragment has an amino acid sequence represented by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 in the sequence listing below. Or a nucleotide sequence encoding a non-A non-B hepatitis virus antigen expressed in whole or in part. The nucleotide sequence includes all sequences based on the degeneracy of the genetic code. Specific examples of such a nucleotide sequence include all or a part of the sequence from nucleotide numbers 1 to 700 shown in SEQ ID NO: 1, all or a part of the sequence from nucleotide numbers 1 to 909 shown in SEQ ID NO: 2, All or part of the sequence from nucleotide numbers 1 to 852 shown in SEQ ID NO: 3, all or part of the sequence from nucleotide numbers 1 to 819 shown in SEQ ID NO: 4, from nucleotide number 3 shown in SEQ ID NO: 5 All or a part of the sequence from nucleotide numbers 1 to 594 shown in SEQ ID NO: 6, all or a part of the sequence from nucleotide numbers 2 to 1141 shown in SEQ ID NO: 7, All or part of the sequence from nucleotide numbers 1 to 1134 shown in SEQ ID NO: 8, shown in SEQ ID NO: 9 All or a part of the sequence from nucleotide numbers 2 to 1663, all or a part of the sequence from nucleotide numbers 2 to 667 shown in SEQ ID NO: 10, and All or part, all or part of the sequence from nucleotide number 2 to 1174 shown in SEQ ID NO: 12, all or part of the sequence from nucleotide number 2 to 1057 shown in SEQ ID NO: 13, shown in SEQ ID NO: 14 Or all or a part of the sequence from nucleotide numbers 2 to 646.
[0037]
The present invention also provides an expression vector in which the nucleic acid sequence has been introduced into a cloning site in a vector located downstream of a promoter. Further, the present invention provides a host cell containing the expression vector.
[0038]
As the vector, a virus (eg, vaccinia virus, baculovirus, etc.) is used in addition to a conventional vector such as a plasmid or a phage. Whether a recombinant peptide or polypeptide obtained by DNA expression has a sugar chain structure determines the type of promoter and host that can be used. That is, when the recombinant peptide or polypeptide does not contain a sugar chain structure, a prokaryote such as Escherichia coli, Bacillus subtilis, or actinomycete can be used as a host, and a promoter such as tryptophan synthase can be used. Operon (trp), lactose operon (lac), lambda phage PL, PR and the like can be used. In this case, it would generally be obtained as a fusion with another peptide. On the other hand, when the recombinant peptide or polypeptide contains a sugar chain structure, the host includes, for example, eukaryotes such as yeast, plant cells, insect cells, and animal cells. Promoters for glycolytic enzymes such as 3-phosphoglycerate kinase and enolase, promoters for alcohol dehydrogenase, and viral promoters usable in mammalian cells such as polyoma virus, adenovirus, simian virus SV40, vaccinia virus, Examples include promoters derived from megalovirus and the like.
[0039]
The vector may further optionally contain a marker sequence (for example, ampicillin, tetracycline resistance gene, etc.) that enables phenotypic selection of the transformed cells, an origin of replication, a terminator, a ribosome binding site, and the like.
[0040]
The present invention further provides a method for producing a recombinant non-A non-B hepatitis virus antigen peptide or polypeptide. This method is, specifically, a step of constructing a replicable expression vector capable of expressing the above-described nucleic acid fragment of the present invention in a suitable host cell,
Step of obtaining a transformant by introducing the expression vector into a host cell,
Culturing the transformant under conditions capable of expressing the nucleic acid fragment to express the recombinant peptide or polypeptide, and
Recovering the recombinant peptide or polypeptide
Is included.
[0041]
Culture conditions for the transformant are determined depending on the host cell to be used, and a medium capable of growing, a culture temperature, a culture time, and the like are appropriately selected. Purification of the recombinant peptide or polypeptide from the culture can be carried out by conventional techniques such as ultrasonic crushing of cells, solubilization extraction, ammonium sulfate fractionation, and various types of chromatography.
[0042]
As used herein, the term "recombinant peptide or polypeptide" refers to a peptide or polypeptide itself obtained by expressing a DNA encoding a non-A non-B hepatitis virus antigen incorporated in an expression vector, or another peptide or polypeptide. And a fusion peptide or polypeptide.
[0043]
The present invention also includes a recombinant peptide or polypeptide obtained by the above method. Such peptides or polypeptides can also be chemically synthesized using conventional peptide synthesis techniques, as will be apparent to those skilled in the art.
[0044]
After the recombinant polypeptide obtained by the present invention was subjected to SDS-polyacrylamide electrophoresis, it was reacted with normal human serum and non-A non-B hepatitis patient serum by Western blotting, as shown in FIGS. 4 and 5. In addition, this recombinant polypeptide reacted only with the serum of a non-A non-B hepatitis patient. Therefore, the recombinant polypeptide is an antigen specific to non-A non-B hepatitis virus and can be used for diagnosis of non-A non-B hepatitis and detection of non-A non-B hepatitis virus.
[0045]
【Example】
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.
[0046]
Example 1
Detection of HCV (# S14) gene by RT-PCR
(4) As a method for cloning the HCV gene from the plasma of a chronic non-A non-B hepatitis patient, the HCV gene was cloned by using the RT (reverse transcriptase) -PCR method, which can be cloned with a small amount of plasma.
[0047]
First, 6 M GTC solution (6 M guanidine thiocyanate, 37.5 mM sodium citrate, 0.75% sarkosyl, 0.2 M mercaptoethanol) was added to 100 μl of a single chronic phase non-A non-B hepatitis plasma (# S14). 200 μl and 1 μl of yeast t-RNA (10 mg / ml) are added and stirred. Further, 20 μl of 3M sodium acetate (pH 5.2), 30 μl of TE-saturated phenol (pH 7.5 to 8.0), and 70 μl of chloroform / isoamyl alcohol (49: 1) were added, mixed quickly, stirred for 10 seconds, and then placed on ice. Let stand for 15 minutes. Centrifuge in a centrifuge at 15000 rpm for 20 minutes at 4 ° C. Take the aqueous layer, mix with an equal volume of isopropyl alcohol and place at -20 ° C for 1 hour or more. This is centrifuged at 15000 rpm for 20 minutes at 4 ° C. to precipitate. The precipitate is dissolved in 100 μl of 4 M GTC (6 M GTC diluted with sterile water), mixed with an equal volume of isopropyl alcohol, and allowed to stand at −20 ° C. for 1 hour or more. Centrifuge at 15000 rpm for 20 minutes at 4 ° C to obtain a precipitate. After washing with 1 ml of 70% ethanol, it was air-dried at room temperature, dissolved in 10 μl of sterilized water, and used as RNA.
[0048]
For cDNA synthesis, 10 μl of RNA is dispensed into a siliconized tube (0.5 ml), heated at 70 ° C. for 3 minutes, and rapidly cooled on ice. Next, 1 μl of RNase inhibitor (Takara Shuzo) (50 units / μl), 1 μl of dNTP (20 mM each), 100 mM DTT, 5 × RT buffer (250 mM Tris-HCl (pH 8.5), 375 mM KCl, 15 mM MgCl 2) 4 μl, random oligo Add 1 μl of hexamer primer (100 pmol / μl) and 1 μl of reverse transcriptase (BRL) (200 units / μl), and adjust to a total of 20 μl with sterile water. After reacting at 42 ° C for 2 hours, the mixture was heated at 94 ° C for 5 minutes to inactivate the enzyme. PCR was performed using this cDNA. PCR used a two-step method to increase the amplification sensitivity and specificity of the detected DNA. That is, first, the first PCR is performed using two kinds of primers (1st @ step @ PCR). Next, a second PCR is performed using two kinds of primers present inside the DNA sequence of the PCR product (2nd step PCR).
[0049]
C14-1, C14-2, C14-3, C14-4, C14-5, C14-6, C14-7, C14-8, C14-9, C14-10, Primers were synthesized for 12 regions of the C14-11 region and the C14-12 region, and used in the two-step method. The PCR primers used are described below. In addition, each area | region is a sequence (J. @ Virol. @ 65, {1105-1113} (1991); @ Proc. @ Natl. \ Acad. \ Sci. \ USA \ 87, 9552-9528 (1990); \ Virus \ Genes 5: 3, \ 243-259. (1991); {J. {General} Virol. {72, 2697-2704} (1991)). 1 and 2 show the positional relationship between the respective amplified regions and the previously reported sequence (HC-J6).
[0050]
For the C14-1 region, primers 14-1: 5′-CGATTGGGGGCGA-3 ′ and 14-2: 5′-TTGCAAAAATTAACCCCGTCCTCCAG-3 ′ were used for the first PCR, and primers 14-1 and 14- were used for the second PCR. 3: 5'-CATGAGGTCCGGCGAAGCCGC-3 'was used.
[0051]
For the C14-2 region, the first PCR uses primers 14-8: 5′-CACCAATGGCAGTTTGCCACATCAAC-3 ′ and 14-9: 5′-GGACTACCCCGACCCTTGATGTACCA-3 ′, and the second PCR uses primers 14-10: 5′-CTGTTCTCACCACCCACAGCTTCACAC. -3 'and 14-11: 5'-GCGTGCAAGACGACCAACTTCTCTA-3' were used.
[0052]
For the C14-3 region, the first PCR uses primers 14-12: 5'-GAGCGGGAGACAGCTGCTTGCGGGGA-3 'and 14-13: 5'-ATAGGTGGAGGTACGTGATGGG-3', and the second PCR uses primers 14-14: 5'-TTCCCCTGTCCCCCCCGA. -3 'and 14-13 were used.
[0053]
For the C14-4 region, the first PCR uses the primers 14-4: 5'-TGGGCAGGATGGCTCCTGTC-3 'and 14-5: 5'-GCCGTGTGTAGGTGACCAGTC-3', and the second PCR uses the primers 14-6: 5'-TGGGTAAGGTCATCGATACC. -3 'and 14-5 were used.
[0054]
For the C14-5 region, the first PCR uses primers 14-15: 5'-CTGGTAGTGGAAAGAGCACCAAAAGT-3 'and 14-16: 5'-TGCATGCACGTGGCGATGTTA-3', and the second PCR uses primers 14-17: 5'-TCGCGCTATGCCGCTCAGGG. -3 'and 14-18: 5'-GTCAGGGGTAACCTCGTTGGGTA-3' were used.
[0055]
Furthermore, the PCR primers shown below were used for the C14-6, C14-7, C14-8, C14-9, C14-10, C14-11 and C14-12 regions.
[0056]
[Table 15]

(N indicates a mix of G, A, T, and C).
[0057]
The PCR conditions were as follows: 20 μl of the above cDNA synthesis reaction solution in a 0.5 ml tube, 8 μl of 10 × PCR buffer (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl 2, 0.1% gelatin), 1st Add 2 types of step primers (75 pmole each), 8 μl of 2 mM dNTP, and make up to 100 μl with sterile water. After heating at 94 ° C. for 10 minutes, 1 μl (5 units) of Ampli {Taq} (Perkin-Elmer-Cetus) is added, and after stirring, mineral oil is overlaid and lightly centrifuged. The PCR reaction was performed for 30 cycles under the conditions of denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, and extension at 72 ° C. for 2 minutes. Next, 10 μl of the 1st PCR reaction completion solution and 9 μl of 10 × PCR buffer are added to a new 0.5 ml tube, and 2 kinds of 2nd step primers (75 μmol each), 9 μl of 2 mM dNTP, and 100 μl with sterile water. After heating at 94 ° C. for 10 minutes, adding Ampli Taq 1 μl (5 units) and stirring, overlay mineral oil, lightly centrifuge, and perform 2nd PCR under the above conditions. After the reaction, 10 μl of the reaction solution was subjected to agarose gel electrophoresis, and a specifically amplified DNA fragment was detected.
Cloning of PCR product (HCV # S14 DNA fragment) and determination of nucleotide sequence
(4) It was considered that the HCV gene might be easily mutated during replication. Therefore, a vector (pBM) obtained by modifying pBR322 (Sutchlife, J. G., Cold Spring Harbor Symposium, 43, 77-90 (1979)) was used as a vector in order to minimize artificial mutations that occur during cloning. . pBM deletes the sequence between the EcoR @ V site and the Bal @ I site of pBR322 with a restriction enzyme, and pUC119 (Vieria, @J., @Messing, @J., \ Methods \ in \ Enzymology,) between the EcoR \ I site and the Hind \ III site. 153, {3-11} (1987)) from the EcoR I site to the Hind III site (ΔpBR MCS). Next, the sequence between the Vsp I site and the Sca I site of pBR322 was replaced with the sequence between the VspI site and the Sca I site of pUC119, and the Pst I site between them was deleted to prepare a 3122 bp full-length vector (FIG. 3). .
[0058]
The PCR reaction solution in which HCV DNA was detected was mixed with an equal volume of chloroform / isoamyl alcohol (24: 1), centrifuged, and the aqueous layer was transferred to a 0.5 ml tube. Sodium acetate (pH 5.2) and a 2-fold amount of ethanol were added to perform ethanol precipitation. The precipitate was dissolved in 300 μl of 10 mM Tris-HCl-1 mM @ EDTA (pH 7.4) (TE), and subjected to centrifugal filtration with Ultrafree C3TK (Nippon Millipore Limited) to remove residual primers and desalinate. The processing solution was sterilized by adding 2 μl of 10 × T4 DNA polymerase buffer (30 mM Tris acetate, 0.66 M potassium acetate, 0.1 M magnesium acetate, 5 mM DTT, 1 mg / ml BSA), 2 μm dNPT 1 μl, and 4 units of T4 DNA polymerase (Takara Shuzo). The volume was adjusted to 20 μl with water and reacted at 12 ° C. for 15 minutes. After the reaction, extraction was performed once each with an equal amount of phenol / chloroform (25:24) and chloroform / isoamyl alcohol (24: 1), and the aqueous layer was precipitated with ethanol. The precipitate was washed with 75% ethanol, air-dried, and 4 μl of 10 × imidazole buffer (0.5 M imidazole hydrochloride (pH 6.4), 0.18 M magnesium chloride, 50 mM DTT), 10% of 24% polyethylene glycol 6000 10 μl, and 10 mM 0.5 μl of ATP and 20 units of T4 DNA kinase (Takara Shuzo) were added to make up to 40 μl with sterilized water, and reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end. The enzyme was inactivated by chloroform / isoamyl alcohol treatment, and the aqueous layer was precipitated with ethanol and washed with 75% ethanol. The precipitate was isolated from the DNA by low-melting point agarose gel electrophoresis, extracted twice with TE-saturated phenol, and the DNA fragment was precipitated with ethanol, washed with 75% ethanol, dissolved in 10 μl of sterilized water, and 1 μl of the agarose gel was separated. After electrophoresis, the amount of the DNA fragment was determined.
[0059]
The DNA fragment obtained here is cut in advance with the restriction enzyme SmaｍI, and a ligation reaction is carried out with a pBM vector whose 5 ′ end has been dephosphorylated by alkaline phosphatase treatment.
[0060]
pBM (20 μl) was reacted at 30 ° C. for 90 minutes in 50 μl of a restriction enzyme reaction solution (10 mM Tris-HCl (pH 8.0), 7 mM MgCl 2, 20 mM KCl, Sma I (Takara Shuzo) 80 units), and 68 ° C., 15 minutes After heating, ethanol precipitates. The precipitate is washed with 75% ethanol, air-dried, and 5 μl of 10 × alkaline phosphatase buffer (100 mM {Tris-HCl (pH 8.3), 1 mM {ZnCl 2}, 10 mM {MgCl 2}), 1 unit of alkaline phosphatase (from bovine small intestine; Takara Shuzo) Dephosphorylation was carried out by adding sterilized water to 50 μl and reacting at 37 ° C. for 1 hour. After adding 0.5 μl of 500 mM EDTA (pH 7.5) and 2.5 μl of 10% SDS, protease K was further added to a final concentration of 50 μg / ml, and reacted at 56 ° C. for 30 minutes to inactivate the enzyme. The vector was isolated by low-melting-point agarose gel electrophoresis, extracted twice with TE-saturated phenol, precipitated with ethanol, washed with 75% ethanol, air-dried, and dissolved in 50 μl of sterilized water. 1 μl of the resultant was subjected to agarose gel electrophoresis, the amount of the vector was determined, and a SmaΔI cloning vector having a final concentration of 0.1 μg / ml was obtained.
[0061]
The phosphorylated DNA fragment was added at a molar ratio of 15-fold to 20-fold with respect to 25 ng of the SmaΔI cloning vector, and 2 μl of 10 × ligation buffer (0.66 M Tris-HCl (pH 7.6), 50 mM MgCl2, 50 mM DTT) Then, 2 μl of 10 mM hexamine cobalt chloride, 2 μl of BSA (1 mg / ml), 1 μl of 10 mM ΔATP, and 350 units of T4 DNA ligase (Takara Shuzo) were added to make up to 20 μl with sterilized water, and a ligation reaction was performed at 16 ° C. overnight. To this reaction solution, 0.5 μl of tRNA (10 mg / ml) was added, and after ethanol precipitation, the precipitate was washed with 75% ethanol. The precipitate was dissolved in 10 μl of sterilized water, and half of the precipitate was used for E. coli JM109 strain or SCS1. The strain was transformed. The susceptible Escherichia coli strain (competent cell) used for the transformation was prepared based on the previous report (J. Mol. Biol., 166, 577 (1983)).
[0062]
The transformant was cultured on an LB-Amp plate (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar, 50 μg / ml ampicillin) overnight, and then placed on the plate. The appearing colonies were cultured in 15 ml tubes each containing 3 ml of LB-Amp, and 1.5 ml of the culture solution was centrifuged to collect the cells. Minipreparation of plasmid DNA (Maniatis et al., Molecular Cloning: A Laboratory Manual, 1982). Was performed to prepare 15 μl of a DNA solution. 2 to 3 μl of the mixture was reacted at 37 ° C. for 1 hour in 10 μl of restriction enzyme EcoR I and Hind III 4 units each in a reaction buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2, 1 mM DTT, 100 mM NaCl). After that, agarose gel electrophoresis was performed to confirm the size of the inserted DNA fragment.
[0063]
Each of the 12 regions has a C14-1 region of approximately 710 bp, a C14-2 region of approximately 950 bp, a C14-3 region of approximately 850 bp, a C14-4 region of approximately 600 bp, a C14-5 region of approximately 1200 bp, and a C14-6 region of approximately 12 bp. 1134 bp, the C14-7 region is about 1664 bp, the C14-8 region is about 667 bp, the C14-9 region is about 1120 bp, the C14-10 region is about 1174 bp, the C14-11 region is about 1057 bp, and the C14-12 region is about 648 bp. DNA fragments were each confirmed.
[0064]
The nucleotide sequences of the obtained 12 types of DNAs were further determined by the dideoxy termination method of Sanger et al. (Science, {214, {1205-1210} (1981)). The respective region clones used for the DNA base sequence determination were C14-1, C14-2, C14-3, C14-4, C14-5, C14-6, C14-7, C14-8, C14-9. , C14-10, C14-11 and C14-12. In addition, the determined nucleotide sequence of the gene and the amino acid sequence deduced therefrom are C14-1 as SEQ ID NO: 1, C14-2 is SEQ ID NO: 2, C14-3 is SEQ ID NO: 3, C14-4 is SEQ ID NO: 6, C14-5 is SEQ ID NO: 7, C14-6 is SEQ ID NO: 8, C14-7 is SEQ ID NO: 9, C14-8 is SEQ ID NO: 10, C14-9 is SEQ ID NO: 11, C14-10 is SEQ ID NO: 12, C14- 11 is shown as SEQ ID NO: 13, and C14-12 is shown as SEQ ID NO: 14. The above plasmids were used as transformants. C14-1 was No. 13029 from CHIKKEN, C14-2 was 13030, C14-3 was 13031, C14-4 was 13032, C14-5. Was issued on June 24, 1992 as No. 13033, and C14-6 was FERM {P-13592}, C14-8 was P-13593, C14-9 was P-13594, and C14-10 was P-13595 # and C14-11 were deposited with the P-13596 # and C14-12 with P-13597 #, respectively, on April 9, 1993 with the Institute of Biotechnology and Industrial Technology.
[0065]
Example 2
Detection of HCV (# 4) gene by RT-PCR
In the same manner as described in Example 1 above, RT-PCR of the HCV (# 4) gene from a single chronic non-A non-B hepatitis patient plasma different from Example 1 was performed, and C4-1 and C4-1 An amplified DNA fragment in the C4-2 region was detected.
[0066]
The primers used are shown below.
[0067]
For the C4-1 region, the first PCR uses primers 4-1: 5'-ATGGAGACTAACTCATCAC-3 'and 4-2: 5'-ACTGTTGCCGATGCCCAAGAT-3', and the second PCR uses primers 4-3: 5'-TACTTCTAGGACCGCCGCAT. -3 'and 14-13: 5'-ATAGGTGGAGGTACGTGATGGG-3' were used.
[0068]
For the C4-2 region, the first PCR uses primers 4-4: 5′-TGGAGCGTATATGTCCAAGG-3 ′ and 4-5: 5′-GACATGCATGCCATCATAGTGTA-3 ′, and the second PCR uses primers 4-4 and 4-6: 5′-CACATTTGGTCCCACGATGGG-3 ′ was used.
Cloning of PCR product and determination of nucleotide sequence
At the time of cloning, pUC119 was used as a vector, the gene fragment amplified by PCR using the SmaΔI site and the above primers was cloned by the method described in Example 1, and the nucleotide sequence was determined. The respective region clones used for the DNA sequence determination were named C4-1 and C4-2. The determined nucleotide sequence of the gene and the amino acid sequence deduced therefrom are shown in SEQ ID NO: 4 for C4-1 and in SEQ ID NO: 5 for C4-2. The above plasmids have been deposited as transformants on June 24, 1992, C4-1 as strain No. 13027 and C4-2 as strain No. 13028.
[0069]
Example 3
Expression of HCV (# S14) -derived gene using E. coli (Part 1)
a) Construction of expression plasmid
Using the DNA of clone C14-1 obtained in Example 1 above, PCR was performed under the PCR conditions of Example 1 using primers B1: 5′-CATGAGCATAAATCTCAAACCTCAAAG-3 ′ and B2: 5′-ATCTGCAGTTTATAGGGTGTCGATGACCTTACCC-3 ′. Then, a DNA fragment of about 380 bp was amplified. The entire amount of the PCR reaction solution was treated with chloroform / isoamyl alcohol (24: 1) according to the method of Example 1, and the aqueous layer was precipitated with ethanol, dissolved in 300 μl of TE, centrifugally filtered, and the remaining primer was removed and desalted. After treatment with T4 DNA polymerase, the 5 ′ end was phosphorylated by T4 DNA kinase treatment. The obtained DNA fragment was digested in a reaction buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 100 mM NaCl) by adding 20 units of PstII, and subjected to low melting point agarose gel electrophoresis. DNA was isolated from agarose gel, extracted twice with TE-saturated phenol, precipitated with ethanol, dissolved in 10 μl of sterilized water, and purified as a DNA fragment of about 380 bp. The DNA fragment obtained here was digested with the restriction vector PKK223-3 (Pharmacia) using the restriction enzyme Pst I in advance under the conditions described above, and further cut with the restriction enzyme Sma I under the conditions described in Example 1 to obtain an alkaline A ligation reaction was carried out with T4 DNA ligase under the conditions of Example 1 and 25 ng of the vector whose 5 ′ end had been dephosphorylated by phosphatase treatment, followed by transformation using Escherichia coli JM105 strain.
[0070]
The transformed bacteria appeared on the plate after overnight culture on an LB-Amp plate (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar, 50 μg / ml ampicillin). Each colony was cultured in a 15 ml tube containing 3 ml of LB-Amp, and 1.5 ml of the culture was centrifuged to collect the cells, followed by mini-preparation of plasmid DNA ({Maniatis et al, Molecular Cloning: A Laboratory Manual, 1982)}. , 15 μl of a DNA solution were prepared. 2-3 μl of the mixture was reacted at 37 ° C. for 1 hour in 10 μl of a reaction buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2, 1 mM DTT, 100 mM NaCl) in 4 units of each of restriction enzymes EcoR I and Pst I. Thereafter, agarose gel electrophoresis was performed to obtain a clone into which a DNA fragment of about 380 bp was inserted.
b)Confirmation of expression by Western blotting and reaction with non-A, non-B hepatitis patient serum
(4) The above Escherichia coli clone was pre-cultured in 3 ml of LB-Amp medium at 37 ° C. for 3 hours, and then 50 μl thereof was inoculated into 5 ml of fresh LB-Amp medium and cultured at 37 ° C. for 2 hours. IPTG (Wako Pure Chemical Industries) was added to the culture solution to a final concentration of 2 mM, and the mixture was further cultured at 37 ° C. for 3 hours. After 1.5 ml of the culture solution was centrifuged at 13000 rpm for 2 minutes, the cells were collected, washed with 1 ml of TE, centrifuged at 13000 rpm for 2 minutes, and collected again. 50 μl of sterile water and 50 μl of 2 × sample buffer (100 mM @ Tris-HCl (pH 6.8), 20% glycerol, 10% SDS, 5% @ 2-mercaptoethanol, 0.2% bromphenol) Blue) was added thereto, and the mixture was suspended and mixed. The suspension was boiled at 100 ° C. for 5 minutes, and then subjected to ultrasonic treatment under ice-cooling.
[0071]
30 μl of the above sample was subjected to SDS-polyacrylamide gel electrophoresis using MINI PROTEAN II Dual SlabCell (Biorad) at 15 mA for 1.5 hours according to the method of Laemmli (Nature, 227, 680 (1970)). After the electrophoresis, the gel was taken out, and a PVDF membrane (Millipore) was adhered to the gel, followed by transfer at 250 mA for 1.75 hours using MINI TRANS BLOT Electrophoretic Transfer Cell (Biorad). After the transfer, the membrane was immersed in a buffer solution I '(10 mM Na-phosphate (pH 7.0), 1% BSA, 0.15 M NaCl, 2.5 mM EDTA) containing 5% skim milk and 2% BSA, and then incubated at room temperature for 2 hours. Time blocked. The blocked membrane was added to a serum sample diluted 40-fold with a buffer solution I 'containing 5% skim milk and 2% BSA, and reacted at room temperature for 4 hours. After the reaction, the membrane was washed three times with a buffer II (10 mM Na-phosphate (pH 7.0), 0.15 M NaCl, 0.05% Tween 20), and then adjusted to 100 mu / ml with a buffer I ′ containing 2% skim milk. The diluted anti-human IgG-POD-labeled antibody solution (goat antibody) was added and reacted at room temperature for 30 minutes. After the reaction, the membrane was taken out and washed five times with Buffer II. The washed membrane is immersed in a coloring solution (20 mM {Tris-HCl (pH 7.5), 0.5 M NaCl, 0.05% 4-chloro-1-naphthol, 0.018% H2 {O2}, 16.7% methanol) and room temperature. For 15 minutes.
[0072]
The results are shown in FIG. FIG. 4 shows the results of Western blots performed on 5 sera of non-A non-B chronic hepatitis patients (No. 1 to No. # 5)} and 5 sera of healthy subjects (No. # 6 to No. # 10). However, only in the serum of non-A non-B hepatitis patients, a strong positive reaction was detected in all, and the expressed antigen was useful for diagnosis of non-A non-B hepatitis patients and detection of non-A non-B hepatitis virus carriers. It was shown that there is.
[0073]
Example 4
Expression of HCV (# S14) -derived gene using E. coli (Part 2)
a) Construction of expression plasmid
The DNAs of the clones C14-3 and C14-5 obtained in Example 1 were digested with restriction enzymes EcoRI, PstI and EcoRI, respectively, in a buffer (Takara Universal Buffer H), and then subjected to agarose gel electrophoresis to about 930 bp and about 930 bp, respectively. A 950 bp DNA fragment was isolated, treated with TE-saturated phenol and chloroform, precipitated with ethanol, and purified by dissolving in 25 μl of sterile water. Each of the purified DNAs was digested with the restriction enzyme ScaI in the above-mentioned buffer solution, DNAs of about 780 bp and 920 bp were isolated by agarose gel electrophoresis, and treated with TE-saturated phenol and chloroform and purified by ethanol precipitation. The two purified DNAs and the vector pBluescript previously digested with the restriction enzyme EcoRI in the above buffer were mixed, and a ligation reaction was carried out with T4 DNA ligase, followed by transformation using Escherichia coli JM109 strain.
[0074]
The transformed bacteria were cultured on an LB-Amp plate (1% bactotryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar, 50 μg / ml ampicillin) overnight, and then colonies appeared on the plate Was cultured in a 15 ml tube containing 3 ml of LB-Amp, and 1.5 ml of the culture was collected by centrifugation, followed by mini-preparation of plasmids (Maniatis et al, Molecular Cloning: A Laboratory Manual, 1982). 20 μl of a DNA solution was prepared. 4 μl of the prepared DNA solution was digested with EcoRI in a buffer solution (Takara Universal Buffer H) and subjected to agarose gel electrophoresis to obtain a clone (28-14D) into which a DNA fragment of about 1700 bp was inserted. Using the DNA of this clone 28-14D, PCR was carried out using primer F2: 5'-CAGAATTCATGGGAAAACTCGGACATCGCC-3 'and primer R: 5'-CACTGCAGTTATGAGACAGCGTCTTTGAGGAC-3'. In the PCR reaction, 1 μl of the above DNA was added to 5 μl of a 10 × PCR buffer (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl 2, 0.1% gelatin), primer F2, R (240 pM each), 25 mM dNTP 0.1 μl. Add 2 μl, 0.2 μl (1 unit) of Taq @ polymerase (Boehringer), make up to 50 μl with sterile water, stir, overlay with mineral oil, denature at 94 ° C. for 0.5 min, annealing at 55 ° C. for 0.5 min, elongate 44 cycles were performed at 72 ° C. for 1 minute. After the reaction, the whole amount of the reaction solution was treated with TE-saturated phenol and chloroform, and the aqueous layer was precipitated with ethanol, dissolved in 40 μl of sterilized water, and added with 5 μl of a buffer solution (Takara \ Universal \ Buffer \ H), 20 units of EcoRI restriction enzyme and 20 units of PstI. And digested. After digestion, a DNA fragment of about 860 bp was isolated by 1.5% agarose gel electrophoresis, treated with TE-saturated phenol and chloroform, precipitated with ethanol, and dissolved in 5 μl of sterilized water to obtain purified DNA. 1 μl of the DNA fragment obtained here was digested with the expression vector pKK223-3 (Pharmacia) in advance with the restriction enzymes EcoRI and PstI, mixed with 1 μl purified under the above conditions, ligated with T4 DNA ligase, and Transformation was performed using the JM109 strain.
[0075]
The transformant appeared on the plate after overnight culture on an LB-Amp plate (1% bactotryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar, 50 μg / ml ampicillin). Each colony was cultured in a 15 ml tube containing 3 ml of LB-Amp, and 1.5 ml of the colony was collected by centrifugation, followed by mini-preparation of plasmid (Maniatis et al, Molecular Cloning: A Laboratory Manual, 1982). , 20 μl of a DNA solution was prepared. 4 μl of the prepared DNA solution was digested with EcoRI and PstI in a buffer solution (Takara Universal Buffer H) and subjected to agarose gel electrophoresis to obtain a clone into which a DNA fragment of about 860 bp was inserted.
b) Confirmation of expression by Western blotting and reaction with non-A non-B hepatitis patient serum
The E. coli clone was pre-cultured in 3 ml of LB-Amp medium at 37 ° C. for 3 hours, and 50 μl of the clone was inoculated into 5 ml of fresh LB-Amp medium and cultured at 37 ° C. for 2 hours. IPTG (Wako Pure Chemical Industries) was added to the culture solution to a final concentration of 2 mM, and the mixture was further cultured at 37 ° C. for 3 hours. After 1.5 ml of the culture solution was centrifuged at 13,000 rpm for 2 minutes to collect the cells, the cells were washed with 1 ml of TE, centrifuged at 13000 rpm for 2 minutes, and collected again. 50 μl of sterilized water and 50 μl of 2 × sample buffer (100 mM Tris-HCl (pH 6.8), 20% glycerol, 10% SDS, 5% 2-mercaptoethanol, 0.2% bromphenol blue) ) Was added and mixed by suspension. The suspension was boiled at 100 ° C for 5 minutes, and then subjected to ultrasonic treatment under ice-cooling. The sample was repeatedly frozen and thawed at -70 ° C twice to prepare a sample.
[0076]
The sample was subjected to SDS-polyacrylamide gel electrophoresis for 15 hours and 1.5 hours according to the method of Laemmli (Nature, 227, 680 (1970)) using MINI PROTEAN II Dual Slab Cell (Biorad). After the electrophoresis, the gel was cut out, a PVDF membrane (Millipore) was adhered to the gel, and transfer was performed at 250 mA for 1.75 hours using MINI TRANS BLOT Electrophoretic Transfer Cell (Biorad). After the transfer, the membrane was immersed in a buffer solution I ′ containing 5% skim milk and 2% BSA (10 mM Na-phosphate (pH 7.0), 1% BSA, 0.15 M NaCl, 2.5 mM EDTA), and then immersed in a buffer solution at room temperature. Time blocked. The blocked membrane was added to a serum sample diluted 40-fold with a buffer solution I 'containing 5% skim milk and 2% BSA, and reacted at room temperature for 4 hours. After the reaction, the membrane was washed three times with a buffer II (10 mM Na-phosphate (pH 7.0), 0.15 M NaCl, 0.05% Tween 20), and then adjusted to 100 mu / ml with a buffer I ′ containing 2% skim milk. The plate was placed in a diluted anti-human IgG-POD-labeled antibody solution (goat antibody) and reacted at room temperature for 30 minutes. After the reaction, the membrane was taken out and washed five times with Buffer II. The washed membrane is immersed in a coloring solution (20 mM {Tris-HCl (pH 7.5), 0.5 M NaCl, 0.05% 4-chloro-1-naphthol, 0.018% H2 {O2}, 16.7% methanol) and room temperature. For 15 minutes.
[0077]
The results are shown in FIG. FIG. 5 shows the results of Western blots performed on 5 sera of patients with non-A non-B chronic hepatitis (No. 1 to No. # 5)} and 5 sera of healthy subjects (No. # 6 to No. # 10). However, a strong positive reaction was detected in all non-A non-B hepatitis patient sera only, indicating that the expressed antigen is useful for diagnosis of non-A non-B hepatitis patients.
[0078]
【The invention's effect】
According to the present invention, an HCV gene having a homology different from that of a previously reported sequence in base sequence and amino acid sequence has been cloned. The nucleotide sequence obtained by the present invention can be expected to be used for nucleic acid diagnosis in non-A non-B hepatitis patients, and a polypeptide prepared based on the nucleotide sequence can be used for antibody measurement in non-A non-B hepatitis patients. In addition to the above, application to an antigen detection system can be expected by preparing a monoclonal antibody. In particular, in the envelope region, application to a vaccine is also considered, and it is considered to greatly contribute to diagnosis and prevention of HCV infection.
[0079]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the cloning strategy of HCV # S14 and # 4 strains by PCR. In the figure, the upper number attached to each region indicates the position of the nucleotide sequence on HC-J6, and the lower number indicates the name of the PCR primer used. The numbers in parentheses indicate the length (bp) of the DNA fragment.
FIG. 2 shows the cloning strategy of the HCV # S14 strain by PCR. In the figure, the upper number attached to each region indicates the position of the nucleotide sequence on HC-J6, and the lower number indicates the name of the PCR primer used. The numbers in parentheses indicate the length (bp) of the DNA fragment.
FIG. 3 shows the steps of constructing a cloning vector pBM.
FIG. 4 shows that the antigen expressed by the present invention (Example 5) was used for 5 sera of patients with chronic non-A non-B hepatitis (No. 1 to No. 5) and 5 sera of healthy subjects (No. 6 to No. 10). 9 is a photograph showing the result of performing Western blotting using Example 3).
FIG. 5 shows that the antigen expressed by the present invention (Example 5) was obtained for 5 sera of patients with chronic non-A non-B hepatitis (No. 1 to No. 5) and 5 sera of healthy subjects (No. 6 to No. 10). 9 is a photograph showing the result of performing Western blotting using Example 4). Lane M shows molecular weight markers.

Claims (8)

A nucleic acid fragment comprising a nucleotide sequence encoding a non-A non-B hepatitis virus antigen represented by the amino acid sequence shown in SEQ ID NO: 4. The nucleic acid fragment according to claim 1, wherein the nucleotide sequence is a sequence from nucleotide numbers 1 to 819 shown in SEQ ID NO: 4. A nucleic acid fragment comprising a nucleotide sequence encoding a non-A non-B hepatitis virus antigen represented by the amino acid sequence shown in SEQ ID NO: 5. The nucleic acid fragment according to claim 3, wherein the nucleotide sequence is a sequence from nucleotide numbers 3 to 992 shown in SEQ ID NO: 5. An expression vector in which the nucleic acid fragment according to any one of claims 1 to 4 has been introduced into a cloning site in a vector located downstream of a promoter. A host cell comprising the expression vector according to claim 5. A method for producing a peptide or polypeptide of a recombinant non-A non-B hepatitis virus antigen,
A step of constructing a replicable expression vector capable of expressing the nucleic acid fragment according to any one of claims 1 to 4 in a suitable host cell.
Step of obtaining a transformant by introducing the expression vector into a host cell,
A method comprising the steps of: culturing the transformant under conditions capable of expressing the nucleic acid fragment to express the recombinant peptide or polypeptide; and recovering the recombinant peptide or polypeptide. A recombinant peptide or polypeptide obtained by the method according to claim 7.

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