JP3849080B2 - Polypeptide, its production and use - Google Patents

Description

或いは、例えば上記の如く適宜選択されたポリヌクレオチドプローブ又はオリゴヌクレオチドプローブを含有するＨＰ遺伝子検出キットを用いてハイブリダイゼーション法を行えば、試料中のＨＰを特異的且つ高感度に検出することもできる。上記ＨＰ遺伝子としては、ＤＮＡの他、ＲＮＡも含むものである。
以下の実施例において使用される、プラスミド，例えば制限酵素、Ｔ４リガーゼ等の酵素及びその他の物質は市販のものを用い、その使用方法については、常法に従った。また、ＤＮＡのクローニング，宿主細胞の形質転換，形質転換体の培養，得られる培養物からのＨＰ特異的検出用ポリペプチド抗原の採取，精製等における操作方法は、当業界でよく知られているもの、或いは文献等により知ることができる方法である。
以下に参考例、実施例を挙げるが、本発明はかかる記載により何ら限定されるものではない。
【００１６】
【実施例】
〔実施例１〕ＣＰ２抗原をコードするＤＮＡの塩基配列、およびＣＰ２抗原のアミノ酸配列の決定
（１）ＣＰ２抗原のＮ末端アミノ酸配列の決定
ＨＰ（ATCC 43504;Rockville,MD）を１０％のウマ血液を含むＢｒｕｃｅｌｌａ（Difco Laboratories,Detroit,MD）アガープレートに接種し、３７℃で５日間微好気条件下で培養した。生育したＨＰコロニーを４５０ｍｌの１０％ウマ血清を含むＢｒｕｃｅｌｌａ培地に接種し微好気条件下，３７℃で５日間培養した。菌体を遠心分離（6000rpm）で回収し、ＰＢＳ（0.1M Sodium-phosphate,pH7.4, 0.15M NaCl）で洗浄した。菌体を超音波で破砕および可溶化し、プレップセル（バイオラッド社、タンパク分取用電気泳動装置）を用いてＳＤＳ−ＰＡＧＥの約６０ＫＤ付近の画分〔抗ＣＰ２モノクローナル抗体（Sugiyama,T et al, Gastroenterology 101, 77-83, 1991）に反応する画分〕を分取した。これを３回繰り返した後、同ＣＰ２モノクローナル抗体を固定化したセファロース４Ｂカラムに導入した。カラムに吸着したタンパク質画分を０．２Ｍグリシン塩酸緩衝液で溶出し精製ＣＰ２抗原画分を得た。これを更にプレップセルを用いたＳＤＳ−ＰＡＧＥで精製し、電気泳動的に単一となったＣＰ２抗原をアミノ酸配列の分析に用いた。
上記精製ＣＰ２抗原を自動プロテインシーケンサー（ＡＢＩ社、４７７−Ａ型）に掛け、そのＮ末端アミノ酸配列を調べた結果、下記の２０個のアミノ酸配列が明らかになった。
Met Val Asn Lys Asp Val Lys Gln Thr Thr Ala Phe Gly Ala Pro Val Trp AspAsp Asn
この配列は、ＨＰのカタラーゼのＮ末端配列として報告されている配列、Met Val Asn Lys Asp Val Lys Gln Thr Thr Ala Phe Gly Ala Pro Valと完全に一致していた（Westblom, T.U. et al, Eur. J. Clin. Microbiol. Infect. Dis., vol11. No.6, 522-526, June 1992）。
【００１７】
（２）ＨＰゲノムＤＮＡの調製
約０．５ｇ（湿重量）のＨＰ菌体（ATCC43504）を氷冷した１０ｍｌのＰＢＳ（0.1M Sodium-phosphate,pH7.4, 0.15M NaCl）で遠心分離により２回洗浄した後、２．５ｍｌの５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）に懸濁した。これに１２０μｌの１０％ＳＤＳおよび０．５ｍｌのＳＴＥＰ（0.5% SDS,50mM Tris-HCl(pH7.5),0.4M EDTA 1mg/mlプロテアーゼＫ）を加え、５０℃で２時間反応させた。反応後ＴＥ緩衝液（ｐＨ８．０）を２ｍｌ加え、５ｍｌのフェノール／クロロホルムで２回抽出し、タンパク質を除去した。更に等量のエーテルで２回抽出した後、２．５倍量のエタノールを加え、遠心分離によりＤＮＡおよびＲＮＡ混合物を沈澱として回収した。沈澱を乾燥させた後、１ｍｌのＴＥ緩衝液に溶解し、２００μｇのＲＮａｓｅＡを加え３７℃で１時間保温した。等量のフェノール／クロロホルムで２回抽出した後、エーテルで抽出し、エタノール沈澱により約１ｍｇの精製ＤＮＡを得た。
（３）ＣＰ２抗原遺伝子のクローニング
ＣＰ２抗原Ｎ末端の配列情報から推定し合成したオリゴヌクレオチド（１７塩基）をγ³²Ｐ−ＡＴＰで標識してプローブとし、ＨＰゲノムＤＮＡの各種制限酵素（PstI, HindIII, StuI, HaeIII, Sau3AI, SacI, XbaI, BamHI, ApaI, ClaI, XhoIなど）消化産物のサザンブロットハイブリダイゼーション分析を行った。この結果、制限酵素Ｓａｕ３ＡＩでＨＰゲノムＤＮＡを消化することにより、プローブと反応する目的のＤＮＡは、約４００塩基の断片となることが明らかとなった。
２００μｇのＨＰゲノムＤＮＡをＳａｕ３ＡＩで３７℃，１６時間消化し、消化産物のアガロースゲル電気泳動を行い、プローブと反応する約４００塩基のＤＮＡ片が泳動された位置のゲルを分離した。分離したゲルから電気泳動によりＤＮＡを溶出し、フェノール／クロロホルム抽出法で精製した。精製したＤＮＡをエタノール沈澱により回収し、沈澱を７０％エタノールで洗浄した後、乾燥させ約４００塩基のＤＮＡ片群を得た。これをpBLuescriptIISK+のＢａｍＨＩ部位に挿入したもので大腸菌（ＪＭ１０９）を形質転換し、アガープレート上に数千の形質転換体のコロニーを得た。
アガープレート上のコロニーをニトロセルロース膜に転写し、アルカリ変性溶液（1.5M NaCl,0.5M NaOH）に５分間浸し、溶菌およびＤＮＡの変性を行った。次にニトロセルロース膜を中和溶液（1M Tris-HCl pH8.0,1.5M NaCl）に５分間浸し、２×ＳＳＣ（0.3M NaCl,0.03M クエン酸ナトリウム）に５分間浸した後、８０℃で２時間加熱し膜上のＤＮＡを固定した。その膜をハイブリダイゼーション溶液〔6×SSC,0.1% SDS,20mM Tris-HCl(pH8.0),0.125% カゼインナトリウム,2.5mM EDTA,100μg/ml 変性サケ精子DNA〕に４２℃で１時間浸した後、γ³²Ｐ−ＡＴＰで標識した前述のオリゴヌクレオチド（１７塩基）を加え、５０℃から３０℃まで１時間あたり−４℃の温度勾配で反応させた後、さらに３０℃で８時間反応させた。ニトロセルロース膜を溶液から取り出し２×ＳＳＣで１５分間，室温で２回洗浄した後、オートラジオグラフィーを行った。
オートラジオグラフィーで陽性と判定したコロニーを純化し、ＣＰ２抗原のＮ末端側の遺伝子を含む４４０塩基のＤＮＡが挿入されたプラスミドｐＳＫＣ５を得た。
この結果と前述のサザンブロットハイブリダイゼーション分析の結果より得られた制限酵素地図から類推するとＣＰ２抗原のＣ末端側の遺伝子はＰｓｔＩとＳａｕ３ＡＩで断片化される約６００ｂｐのＤＮＡ片に含まれることが考えられた。そこで、残りの領域を取得するために下記のようにクローニング実験を進めた。
２００μｇのＨＰゲノムＤＮＡをＨａｅIIIで部分消化し、消化産物のアガロースゲル電気泳動を行い、プローブと反応する４．５Ｋｂｐ〜３ＫｂｐのＤＮＡ片の画分を分離した。さらにこの画分をＰｓｔＩで３７℃，３時間消化し、消化産物のアガロースゲル電気泳動を行い、プローブと反応する約１．８ＫｂｐのＤＮＡ片を分離した。分離したＤＮＡをＳａｕ３ＡＩで３７℃，１時間消化し、消化産物をプラスミドベクター（pMAL-c2）のＰｓｔＩ−ＢａｍＨＩ切断面を持つものにクローニングした。この結果、ＣＰ２抗原Ｃ末端側の遺伝子を含むＤＮＡ片約６００ｂｐが挿入されたプラスミドｐＭＢＰ２６が得られた。
ｐＳＫＣ５とｐＭＢＰ２６に含まれるＣＰ２抗原遺伝子の塩基配列をDNA Taq Polymeraseを用いたジデオキシ法により決定した。この配列を参考にして作製した２種類の合成オリゴヌクレオチドAAGATGGTTAATAAAGATGTG（配列番号：１の18〜38番目の塩基）及びAAAATCAATGCTGTATTGAGC（配列番号：１の1800〜1820番目の塩基の逆鎖）をプライマーとしてＰＣＲ法（94℃、300秒：94℃、90秒，60℃、120秒，72℃、180秒を30サイクル：72℃、120秒）を行なうことにより、ＣＰ２抗原構造遺伝子を含む断片を増幅した。得られた増幅断片の両末端を平滑化し、リン酸基を付加した後、pTrcHis発現ベクター（Invitrogen社製）の平滑化されたEcoRI切断面を持つものにクローニングし、ＣＰ２抗原構造遺伝子を含むプラスミドｐＴｒｃＣＰ２を得た。
得られたプラスミドｐＴｒｃＣＰ２から、DNA Taq Polymeraseを用いたジデオキシ法により、挿入断片（CP2断片）の塩基配列を決定した。
その配列の解析の結果、ＣＰ２抗原構造遺伝子の配列１５１５ｂｐを含むＨＰゲノムＤＮＡ配列１８２９ｂｐ（配列番号：１）が明らかとなった。これにより５’末端から数えて２１塩基目から始まるＡＴＧを翻訳開始配列とする５０５アミノ酸残基からなるＣＰ２抗原のアミノ酸配列（配列番号：１）が明らかとなった。
この配列の結果をゲネティックス遺伝子解析ソフト（ＳＤＣソフトウエア開発社）のＥＭＢＬ、およびＧｅｎＢａｎｋのデータバンクでホモロジーサーチしたところ、ラットやヒトなどのカタラーゼと５０％以上の相同性を示した。
【００１８】
〔実施例２〕組換えＣＰ２抗原タンパク質の発現
得られた発現ベクターｐＴｒｃＣＰ２をE.coli（JM109）に形質転換したものをＬＢ培地で一晩培養した後、培養液の２０μｌを約３ｍｌのＬＢ培地に植菌し、ＯＤ６００が０．６になるまで培養した。これを２本用意し、内１本にＩＰＴＧを最終濃度１ｍＭになるように加え、更に３時間培養した。培養が終了した２本のサンプル共に遠心分離（5000rpm,10min）後、２倍量のSDS-PAGE loading Buffer〔125mM Tris-HCl(pH6.8), 4%SDS, 0.2% BPB, 20% glycerol〕を加え、３分間煮沸し、ＳＤＳ−ＰＡＧＥを行った。その結果、ＩＰＴＧによって誘導されて発現した組換えＣＰ２抗原蛋白を確認することができた。
【００１９】
〔実施例３〕組換えＣＰ２抗原タンパク質と抗ＣＰ２モノクローナル抗体（Sugiyama,T et al, Gastroenterology 101, 77-83, 1991）との反応
実施例２のＳＤＳ−ＰＡＧＥ終了後、更にセミドライブロッター（ザルトリウス社）を用いて、ポリアクリルアミドゲル中のタンパク質をニトロセルロース膜に転写した。次いで、該ニトロセルロース膜をブロッキング溶液（１％ BSA，20mM Tris-HCl pH7.5，150mM NaCl）中で１時間振蕩した後、抗ＣＰ２モノクローナル抗体（マウス由来）を加え更に１時間振蕩した。ニトロセルロース膜を該溶液から取り出し、ＴＢＳＴ〔20mM Tris-HCl pH7.5，150mM NaCl，0.05％ Tween20（V/V）〕で５分間３回洗浄後、ＴＢＳ（20mM Tris-HCl pH7.5，150mM NaCl）で更に５分間５回洗浄した。次に該ニトロセルロース膜を、ブロッキング溶液で希釈したアルカリホスファターゼ標識抗マウスＩｇＧ抗体溶液に１時間浸した後、該溶液から取り出し、ＴＢＳＴで５分間３回洗浄し、更にＴＢＳで１０分間５回洗浄した。洗浄後、該ニトロセルロース膜を発色液（0.3mg/ml Nitro Blue Tetrazolium,0.15mg/ml 5-Bromo-4-Chloro-3-Indolil Phosphate,100mM Tris-HCl pH9.5,100mM NaCl,5mM MgCl₂ ）中で発色させた。
この結果、組換えＣＰ２抗原タンパク質と抗ＣＰ２モノクローナル抗体が反応することが認められた。
【００２０】
〔実施例４〕異なる一次構造を持つＣＰ２抗原遺伝子の取得
（１）ＨＰ菌体（ATCC43504）からのＣＰ２抗原遺伝子の取得
実施例１（１）でＨＰ菌体（ATCC43504）から調製したＨＰゲノムＤＮＡに対して、実施例１で決定したＣＰ２抗原構造遺伝子を含むＨＰゲノム配列（配列番号：１）に基づいて合成した２種類のオリゴヌクレオチドAAGATGGTTAATAAAGATGTG（配列番号：１の18〜38番目の塩基）及びAAAATCAATGCTGTATTGAGC（配列番号：１の1800〜1820番目の塩基の逆鎖）をプライマーとしてＰＣＲ法（94℃、300秒：94℃、90秒，60℃、120秒，72℃、180秒を30サイクル：72℃、120秒）を行なうことにより、ＣＰ２抗原構造遺伝子を含む断片を増幅した。得られた増幅断片の両末端を平滑化し、リン酸基を付加した後、pBluescriptベクターのSmaI切断面を持つもの、及びpTrcHis発現ベクターの平滑化されたEcoRI切断面を持つものにクローニングし、ＣＰ２抗原構造遺伝子を含むプラスミドｐＢｌｕｅＰＣＲ１及びプラスミドｐＨｉｓＰＣＲ１を得た。
得られたプラスミドｐＢｌｕｅＰＣＲ１から、ディレーションキット（ニッポンジーン社製）を用いて様々な塩基長のディレーションミュータントを作製し、ALFred AutoRead Seqencing kit（ファルマシア社製）を用いたジデオキシ法により、挿入断片（CP2-PCR1断片）の塩基配列を決定した。
その配列の解析の結果、ＣＰ２抗原構造遺伝子の配列１５１５ｂｐを含むＨＰゲノム配列１８０３ｂｐ（配列番号：２）が明らかとなった。これにより、このCP2-PCR1断片中のＣＰ２抗原構造遺伝子は、実施例１で得られたＣＰ２抗原構造遺伝子と、塩基配列が２個、また、それを翻訳した場合のアミノ酸配列が２個異なったものであることが明らかとなった。
CP2-PCR1断片中のＣＰ２抗原構造遺伝子のアミノ酸配列と、実施例１で得られたＣＰ２抗原構造遺伝子のアミノ酸配列とのホモロジーの比較結果を、図１に示す。
（２）ＨＰ臨床菌株からのＣＰ２抗原遺伝子の取得
慢性胃炎患者（24歳、男性）の胃から採取したＨＰ菌体から、実施例１（１）と同様の方法によりＨＰゲノムＤＮＡを調製した。
得られたＨＰゲノムＤＮＡに対して、上記（１）と同様の２種類の合成オリゴヌクレオチドをプライマーとして使用して、ＰＣＲ法（94℃、300秒：94℃、90秒，60℃、120秒，72℃、180秒を45サイクル：72℃、120秒）を行ない、ＣＰ２抗原構造遺伝子を含む断片を増幅した。得られた増幅断片の両末端を平滑化し、リン酸基を付加した後、pBluescriptベクターのSmaI切断面を持つものにクローニングし、ＣＰ２抗原構造遺伝子を含むプラスミドｐＢｌｕｅＰＣＲ２を得た。更に、得られたプラスミドｐＢｌｕｅＰＣＲ２から、BamHI及びXhoIによって挿入断片（CP2-PCR2断片）を切り出し、pTrcHis発現ベクターのBamHI／XhoI切断面を持つものにクローニングして、ＣＰ２抗原構造遺伝子を含むプラスミドｐＨｉｓＰＣＲ２を得た。
得られたプラスミドｐＢｌｕｅＰＣＲ２から、上記（１）と同様に、ディレーションキット（ニッポンジーン社製）を用いて様々な塩基長のディレーションミュータントを作製し、ALFred AutoRead Seqencing kit（ファルマシア社製）を用いたジデオキシ法により、挿入断片（CP2-PCR2断片）の塩基配列を決定した。
その配列の解析の結果、ＣＰ２抗原構造遺伝子の配列１５１５ｂｐを含むＨＰゲノム配列１８１０ｂｐ（配列番号：３）が明らかとなった。これにより、このCP2-PCR2断片中のＣＰ２抗原構造遺伝子は、実施例１で得られたＣＰ２抗原構造遺伝子と、塩基配列が63個、また、それを翻訳した場合のアミノ酸配列が15個異なったものであることが明らかとなった。
CP2-PCR2断片中のＣＰ２抗原構造遺伝子のアミノ酸配列と、実施例１で得られたＣＰ２抗原構造遺伝子のアミノ酸配列とのホモロジーの比較結果を、図１に示す。
尚、図１中、Ｇはグリシンを、Ａはアラニンを、Ｖはバリンを、Ｌはロイシンを、Ｉはイソロイシンを、Ｓはセリンを、Ｔはトレオニンを、Ｄはアスパラギン酸を、Ｅはグルタミン酸を、Ｎはアスパラギンを、Ｑはグルタミンを、Ｋはリジンを、Ｒはアルギニンを、Ｃはシステインを、Ｍはメチオニンを、Ｆはフェニルアラニンを、Ｙはチロシンを、Ｗはトリプトファンを、Ｈはヒスチジンを、Ｐはプロリンを夫々示す。
また、図１中の、上段CP2は、実施例１で得られたＣＰ２抗原構造遺伝子のアミノ酸配列を、中段CP2-PCR1は、実施例４（１）で得られたＣＰ２抗原構造遺伝子（CP2-PCR1）のアミノ酸配列を、また、下段CP2-PCR2は、実施例４（２）で得られたＣＰ２抗原構造遺伝子（CP2-PCR2）のアミノ酸配列を夫々示す。
更に、図１中の＊印は、ＣＰ２抗原構造遺伝子のアミノ酸配列のうち、三者の間で違いが認められるアミノ酸であることを示す。
図１の結果から、ＨＰ由来のＣＰ２抗原構造遺伝子には、同一菌株内での小さな変異や菌株の違いによる大きな変異が生じていること、換言すれば、ＨＰ由来のＣＰ２抗原には幾つかの変異型が存在することが判る。
また、この結果は、Westblom,T.U.らによるカタラーゼ活性陰性を示すＨＰ変異株の報告（Eur. J. Clin. Microbiol. Infect. Dis., Vol 11, No.6, 522-526, June 1992）とも一致する。
【００２１】
〔実施例５〕組換えＣＰ２抗原タンパク質の発現と抗ＣＰ２モノクローナル抗体との反応
実施例４で得られた２種類の組換えベクターｐＨｉｓＰＣＲ１及びｐＨｉｓＰＣＲ２をE.coli（TOP10）に夫々形質転換したものを、実施例２と同様の方法で培養した。培養終了後、実施例２と同様に遠心分離処理し、実施例２と同様の方法で夫々ＳＤＳ−ＰＡＧＥを行い、組換えＣＰ２抗原タンパク質の発現の有無を確認した。更に、実施例３と同様のウェスタンブロッティング法で、組換えＣＰ２抗原タンパク質と抗ＣＰ２モノクローナル抗体との反応性を確認した。
CP2−PCR1組換えタンパク質のＳＤＳ−ＰＡＧＥの結果を図２(ａ)に、また、該ＳＤＳ−ＰＡＧＥを用いた、抗ＣＰ２モノクローナル抗体とのウェスタンブロッティング分析の結果を図２(ｂ)に夫々示す。
また、CP2−PCR2組換えタンパク質のＳＤＳ−ＰＡＧＥの結果を図３(ａ)に、また、該ＳＤＳ−ＰＡＧＥを用いた、抗ＣＰ２モノクローナル抗体とのウェスタンブロッティング分析の結果を図３(ｂ)に夫々示す。
尚、図中の各レーン番号は以下の試料を使用した結果を夫々示す。

また、図２(ａ)及び図３(ａ)中の→印は、組換えＣＰ２抗原タンパク質の位置を示し、また、図２(ｂ)及び図３(ｂ)中の→印は、組換えＣＰ２抗原タンパク質と抗ＣＰ２モノクローナル抗体との反応シグナルの位置を示す。
図２(ａ)及び図３(ａ)の結果から明らかなように、組換えベクターｐＨｉｓＰＣＲ１及びｐＨｉｓＰＣＲ２を夫々含む形質転換体は、何れもＩＰＴＧにより約６０ＫＤの組換えＣＰ２抗原タンパク質（CP2-PCR1組換えタンパク質及びCP2-PCR2組換えタンパク質）を効率よく誘導発現するようになることが判る。
尚、該組換えタンパク質は、何れもヒスチジン残基の６量体を含む融合タンパク質として発現しているので、ＨＰ産生タンパク質中のＣＰ２抗原に比べて、若干その分子量が大きい。
また、図２(ｂ)及び図３(ｂ)の結果から明らかなように、CP2-PCR1組換えタンパク質及びCP2-PCR2組換えタンパク質は、何れもＨＰ由来のＣＰ２抗原に対する抗体、即ち、抗ＣＰ２モノクローナル抗体との反応性を有するものであることが判る。
更に、本発明の組換えＣＰ２抗原タンパク質（約６０ＫＤ）よりも分子量の小さいタンパク質が認められるが、これは、宿主細胞内での分解作用を受けて、該組換えＣＰ２抗原タンパク質が切断されたものと考えられる。これらの組換えタンパク質も、抗ＣＰ２モノクローナル抗体との反応性を有していることから、本発明に於ける配列番号：１、配列番号：２又は配列番号：３で示される配列の一部分の配列も、ＨＰ特異的検出用抗原等として使用可能であることが示唆された。
【００２２】
〔実施例６〕組換えＣＰ２抗原タンパク質と患者血清との反応
実施例４で得られた２種類の組換えベクターｐＨｉｓＰＣＲ１及びｐＨｉｓＰＣＲ２をE.coli（TOP10）に夫々形質転換したものを、５０ｍｌのＳＯＢ培地（2% トリプトファン、0.5% イーストエキストラクト、10mM NaCl、2.5mM KCl、10mM MgCl₂、10mM MgSO₄）で、ＯＤ₆₀₀が0.6になるまで夫々培養し、更にＩＰＴＧが最終濃度１ｍＭになるように加え、２〜５時間培養して、ＩＰＴＧによる誘導発現を行った。培養終了後、遠心分離（5000rpm,10min）処理により菌体を集め、１０ｍｌの溶菌緩衝液（6M Guanidium Hydrochloride、20mM Sodium phosphate、500mM Sodium chloride）に夫々懸濁した。得られた懸濁液を超音波処理して菌体を破砕した後、遠心分離（3000G、15min）処理して沈殿物を除き、得られた上清をProBondレジンカラム（Invitrogen社製：ニッケルカラム）に夫々アプライしてCP2-PCR1及びCP2-PCR2組換えＣＰ２抗原タンパク質をアフィニティー精製した（組換えＣＰ２抗原タンパク質は、ヒスチジン残基の６量体を有する融合タンパク質として発現しているためニッケルにアフィニティーを示す。）。得られた組換えＣＰ２抗原タンパク質を、実施例２と同様の方法でＳＤＳ−ＰＡＧＥを行い、その後セミドライブロッター（ザルトリウス社）を用いて、ポリアクリルアミドゲル中のタンパク質をニトロセルロース膜に転写した。次いで、該ニトロセルロース膜を３ｍｌのブロッキング溶液〔25% block Ace（大日本製薬製）,1% BSA含有するTBS buffer〕中で１時間室温に置いた後、十二指腸潰瘍患者（ＨＰ感染者）の血清を５μｌ加えて、更に１時間反応させた。反応後、該ニトロセルロース膜を取り出してTBST buffer洗浄した後、アルカリホスファターゼ標識された抗ヒトＩｇＧ抗体（カッペル社製）を加えたブロッキング溶液中で１時間反応させた。また対照として、患者血清の代わりに、健常者（ＨＰ非感染者）の血清を用いて同様にウェスタンブロッティング分析を行った。
CP2-PCR1組換えタンパク質とCP2-PCR2組換えタンパク質のＳＤＳ−ＰＡＧＥの結果を図４(ａ)に、また、該ＳＤＳ−ＰＡＧＥを用いた、十二指腸潰瘍患者血清とのウェスタンブロッティング分析の結果を図４(ｂ)に夫々示す。
また、CP2-PCR1組換えタンパク質とCP2-PCR2組換えタンパク質のＳＤＳ−ＰＡＧＥの結果を図５(ａ)に、また、該ＳＤＳ−ＰＡＧＥを用いた、正常（健常者）血清とのウェスタンブロッティング分析の結果を図５(ｂ)に夫々示す。
尚、図中の各レーン番号は以下の試料を使用した結果を夫々示す。

また、図４(ａ)及び図５(ａ)中の→印は、組換えＣＰ２抗原タンパク質の位置を示し、また、図４(ｂ)中の→印は、組換えＣＰ２抗原タンパク質と血清中のＨＰに対する抗体との反応シグナルの位置を、図５(ｂ)中の→印は、組換えＣＰ２抗原タンパク質と血清中のＨＰに対する抗体との反応シグナルが生ずるであろう位置を夫々示す。
図４(ａ)及び図５(ａ)の結果から明らかなように、発現ベクターｐＨｉｓＰＣＲ１及びｐＨｉｓＰＣＲ２を夫々含む形質転換体培養物から、約６０ＫＤの精製組換えＣＰ２抗原タンパク質（精製CP2-PCR1組換えタンパク質及び精製CP2-PCR2組換えタンパク質）が得られることが判る。尚、該組換えタンパク質は、何れもヒスチジン残基の６量体を含む融合タンパク質として発現しているので、ＨＰ産生タンパク質中のＣＰ２抗原及びＨＰ菌体から精製したＣＰ２抗原に比べて、若干その分子量が大きい。
また、図４(ｂ)の結果から明らかなように、得られた精製組換えＣＰ２抗原タンパク質は、何れもＨＰ産生タンパク質中のＣＰ２抗原及びＨＰ菌体から精製したＣＰ２抗原と反応する抗ＣＰ２抗体と反応する性質、即ち、十二指腸潰瘍患者（ＨＰ感染者）血清中の抗ＣＰ２抗体と反応することが判る。また、図５(ｂ)の結果から明らかなように、得られた精製組換えＣＰ２抗原タンパク質は、正常（健常者）血清中の成分とは反応しないことも判る。
以上のことから、本発明の組換えＣＰ２抗原タンパク質を用いれば、患者（ＨＰ感染者）血清中に存在するＨＰに対する抗体を検出し得ること、即ち、ＨＰに感染しているか否かを血清から判定し得ることや、本発明の形質転換体を培養し、その培養物から該組換えＣＰ２抗原タンパク質を分離精製すれば、ＨＰを特異的に検出するための抗原として有用なポリペプチドを容易に且つ大量に得られることが判る。
【００２３】
〔実施例７〕サンドイッチＥＬＩＳＡ法による組換えＣＰ２抗原の評価
９６穴プレートに１０μｇ／ｍｌの濃度の抗ＣＰ２モノクローナル抗体１００μｌずつ分注し、４℃で終夜放置して抗体を固相化した。該プレートは、固相化処理後、ＰＢＳ（１０ｍＭリン酸ナトリウム、０．１５Ｍ塩化ナトリウム）で３回洗浄した。
次いで、該プレートのウェルに４００μｌのブロッキング液（ＰＢＳ、２５％ｂｌｏｃｋＡｃｅ）を分注して室温で６時間放置してブロック処理した後、ＰＢＳで洗浄を３回行なった。
ブロック処理を行ったプレートのウェルに、１０μｇ／ｍｌ濃度の組換えＣＰ２抗原（実施例６で得られたＣＰ２−ＰＣＲ１、ＣＰ２−ＰＣＲ２）溶液を１００μｌずつ分注して４℃で終夜放置した後、ＰＢＳで３回洗浄した。
次に、ブロッキング液で１０００倍希釈した胃潰瘍や胃ガン、十二指腸潰瘍などの患者血清（１０検体）、あるいは正常（健常者）血清（５検体）をそれぞれ１００μｌずつ分注し、４℃で終夜放置した後、ＰＢＳで５回洗浄した。
発色液［ｏ−フェニレンジアミン１０ｍｇ、３０％過酸化水素水１０μｌ、リン酸クエン酸緩衝液（０．１Ｍクエン酸、０．２Ｍリン酸水素ナトリウム、ｐＨ５．０）１０ｍｌ］を１００μｌずつ分注し、室温で５分間反応させた後、１Ｎ硫酸１００μｌで反応を停止させた。
各ウェルの吸光度を、Ｔｍａｘプレートリーダー（モレキュラーデバイス社製）にて計測した（ｍｅａｓｕｒｅ：４９０ｎｍ、ｒｅｆｅｒｅｎｃｅ：６２０ｎｍ）。
ＣＰ２−ＰＣＲ１組換えタンパク質を用いたサンドイッチＥＬＩＳＡ法により、正常（健常者）及び患者血清中の抗ＨＰ抗体に由来する呈色量を測定した結果を表１及び図６（ａ）に夫々示す。
また、ＣＰ２−ＰＣＲ２組換えタンパク質を用いたサンドイッチＥＬＩＳＡ法により、正常（健常者）及び患者血清中の抗ＨＰ抗体に由来する呈色量を測定した結果を表１及び図６（ｂ）に夫々示す。
【００２４】
【表１】

【００２５】
表１及び図６（ａ）及び（ｂ）から明らかな如く、本発明の組換えＣＰ２抗原、ＣＰ２−ＰＣＲ１及びＣＰ２−ＰＣＲ２を用いて抗ＨＰ抗体を測定すると、何れも患者血清と正常（健常者）血清との間で明らかな有意差が生じることが判る。
従って、本発明の組換えＣＰ２抗原を用いてサンドイッチＥＬＩＳＡ法を行った場合でも、血清中の抗ＨＰ抗体を感度良く検出できることが判る。
【００２６】
【発明の効果】
本発明のポリペプチドは、抗ＨＰ抗体を特異的に検出するための抗原として或いはＨＰに対するワクチンとして利用することができる。
また、本発明のＤＮＡを利用すれば、遺伝子工学的手法によりＨＰ特異的検出用抗原として利用できるポリペプチドを容易に且つ大量に製造することができ、更に本発明のＤＮＡを利用してオリゴヌクレオチドプライマー或いはプローブを作製すれば、ＰＣＲ法或いはハイブリダイゼーション法等によりＨＰを検出することも可能である。
本発明の方法によれば、従来取得が困難であったＨＰの特異的検出を可能にするポリペプチド抗原を、容易にかつ大量に取得することができる。
【００２７】
【配列表】

【００２８】
【配列番号：２】

【００２９】
【配列番号：３】

【図面の簡単な説明】
【図１】実施例１で得られたＣＰ２抗原構造遺伝子のアミノ酸配列（配列番号：１）、実施例４（１）で得られたＣＰ２抗原構造遺伝子のアミノ酸配列（配列番号：２）及び実施例４（２）で得られたＣＰ２抗原構造遺伝子のアミノ酸配列（配列番号：３）を比較したものである。図中、＊印は、ＣＰ２抗原構造遺伝子のアミノ酸配列のうち、三者の間で違いが認められるアミノ酸であることを示す。
【図２】（ａ）は実施例５で得られた、CP2-PCR1組換えタンパク質のドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動の結果を示したものである。
（ｂ）は実施例５で得られた、CP2-PCR1組換えタンパク質と抗ＣＰ２モノクローナル抗体とのウェスタンブロッティング分析の結果を示したものである。
【図３】（ａ）は実施例５で得られた、CP2-PCR2組換えタンパク質のドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動の結果を示したものである。
（ｂ）は実施例５で得られた、CP2-PCR2組換えタンパク質と抗ＣＰ２モノクローナル抗体とのウェスタンブロッティング分析の結果を示したものである。
【図４】（ａ）は実施例６で得られた、CP2-PCR1組換えタンパク質とCP2-PCR2組換えタンパク質のドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動の結果を示したものである。
（ｂ）は実施例６で得られた、CP2-PCR1組換えタンパク質又はCP2-PCR2組換えタンパク質と、十二指腸潰瘍患者血清とのウェスタンブロッティング分析の結果を示したものである。
【図５】（ａ）は実施例６で得られた、CP2-PCR1組換えタンパク質とCP2-PCR2組換えタンパク質のドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動の結果を示したものである。
（ｂ）は実施例６で得られた、CP2-PCR1組換えタンパク質又はCP2-PCR2組換えタンパク質と、正常（健常者）血清とのウェスタンブロッティング分析の結果を示したものである。
【図６】（ａ）は、実施例７で得られた、CP2-PCR1組換えタンパク質を用いたサンドイッチＥＬＩＳＡ法により、正常（健常者）血清及び患者血清中の抗ヘリコバクター・ピロリ（ＨＰ）抗体に由来する呈色量を測定した結果を示したものである。
（ｂ）は、実施例７で得られた、CP2-PCR2組換えタンパク質を用いたサンドイッチＥＬＩＳＡ法により、正常（健常者）血清及び患者血清中の抗ＨＰ抗体に由来する呈色量を測定した結果を示したものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel polypeptide, a DNA encoding the same, a recombinant vector containing the DNA, a host cell transformed with the recombinant vector, a method for producing the polypeptide by culturing the host cell, And its uses.
[0002]
[Prior art]
Helicobacter pylori (formerly known as Helicobacter pylori, formerly known as Campylobacter pylori) is a Gram-negative bacilli that was discovered in 1983 as a spiral bacterium detected from chronic gastritis biopsy [Warren, JR & Marshall, B Lancet, i: 1273, 1983].
Since its discovery, the significance of this bacterium in chronic gastritis and duodenal ulcers has attracted attention, and its research has become one of the important biological and medical themes. In particular, the elucidation of the cytotoxic mechanism leading to the disease and the development of new test methods are attracting attention.
Methods for detecting Helicobacter pylori (hereinafter abbreviated as HP) include a culture method, a urease test, a breath test, a histological examination, and a serological method. In particular, the serological method is a method for detecting anti-HP antibodies in serum, and is excellent in that it is noninvasive and can process a large amount of specimens.
Since there are various anti-HP antibodies in serum, the measurement sensitivity and specificity differ depending on the preparation method and type of antigen used for antibody measurement. As a result, the clinical significance of measuring anti-HP antibodies is also A wide variety.
For example, in the detection method [Rathbone, BJLancet, ii: 1217, 1985] using a lysate of whole HP as an antigen for detecting an anti-HP antibody, a false-positive determination is caused by a cross antibody with another Campylobacter genus, On the contrary, there may be false negatives due to interactions between them and denaturation in the antigen preparation process.
Rathbone et al. Have reported that if the antigen is partially purified with an acidic glycine extract from bacterial cells, both specificity and sensitivity will be improved over cell lysates of whole HP cells [Rathbone, BJ: Serological response to Helicobacter pylori European Journal of Gastroenterolology and Hepatology (Eur. J. gastroenterol. Hepatol.), 4 (Supple. 1): S21-S23, 1992]. Furthermore, in the method using urease or the like highly purified by high performance liquid chromatography (HPLC) or the like as the antigen for detection, the specificity increases, but the sensitivity tends to decrease due to the problem of genetic diversity among HP strains. It is reported.
[0003]
In addition, antibodies that correspond to various antigen components of HP were found to be important in relation to the usefulness in serodiagnosis and the pathology of stomach and duodenal diseases. von Wulffen et al. reported that IgG and IgA type antibodies are useful, particularly antibodies against 110 KD and 22 KD antigens appear characteristically in these pathologies [von Wulffen H, Heesemann J, Bulzow GW et al: Journal of Clinical Micro Biology (J. Clin. Microbiol.) twenty four : 716-720,1986]. Goodwin et al. Are conducting ELISA using an acidic glycine extract as an antigen, and the main antigen molecules are 64 KD, 62 KD, and 57 KD, and 84 KD, 33 KD, 28 KD, and 25 KD. Hirschl et al. Say 128 KD antigen is useful [Goodwin CS, Blincow E, Peterson G et al: Journal of Infect. Dis. 155 : 488-494,1987]. Stacey, Newell et al. Have fractionated each antigen component by HPLC, and antibodies against HP-derived urease are useful for serodiagnosis [Stacey AR, Hawtin PR, Newell DG: European Journal of Clinical And Microbiological Infectious Diseases (Eur. J. Clin. Microbiol. Infect. Dis.) 9 : 732-737,1990]. Evans DJ et al. Have set up an ELISA using a high molecular weight cell-associated protein of 600 KD or more as an antigen [Evans Jr DJ, Evans DG, Grahm DX et al: Gastroenterology 96 : 1004-1008, 1989].
On the other hand, Sugiyama et al. Have attempted to detect anti-HP antibodies in blood using HP-derived antigens purified with monoclonal antibodies. As a result, an antigen named CP2 antigen (molecular weight of about 60 KD) has been reported to have excellent HP detection specificity, and its antibody titer is highly correlated with gastritis pathology [Sugiyama, T. et al: Gastro Gastroenterology 101 , 77-83, 1991, and Toshiro Sugiyama et al. 85 1128, 1988].
[0004]
[Problems to be solved by the invention]
In order to actually obtain HP antigens by conventional methods, it has been necessary to isolate them directly from HP cells. However, in order to obtain HP cells, it is necessary to use an expensive liquid medium containing 10% horse serum under microaerobic conditions, or to perform a long culture of about 5 days. Furthermore, it is difficult to separate and purify the target antigen with respect to the whole cells, and it is difficult to obtain a large amount of purified antigen that can be used industrially.
Separation and purification of the CP2 antigen which seems to be particularly useful as a specific antigen was difficult and complicated.
[0005]
[Means for Solving the Problems]
In order to increase the industrial utility of a polypeptide derived from HP that is mainly used as an antigen (CP2 antigen) that enables specific and quantitative examination of HP, the present invention obtains its gene. The amino acid sequence showing the primary structure of the polypeptide, the DNA encoding the same, the vector containing the DNA, and the trait introduced with the vector The purpose is to provide a converter.
As a result of intensive studies to achieve the above object, the present inventors succeeded in cloning the CP2 antigen gene of HP, and succeeded in determining the primary structure of the polypeptide of the present invention that is the CP2 antigen from its base sequence. The present invention has been completed.
That is, the present invention
(1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof,
(2) DNA encoding the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof,
(3) DNA having the base sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof,
(4) a recombinant vector containing the DNA according to (2) or (3),
(5) a cell host transformed with the recombinant vector according to (4),
(6) The method for producing the polypeptide according to (1), wherein the cell host according to (5) is cultured in a medium, and the polypeptide is collected from the obtained culture.
(7) a reagent for measuring anti-Helicobacter pylori antibody, comprising the polypeptide according to (1),
(8) A reagent for detecting a Helicobacter pylori gene having an oligonucleotide or a polynucleotide which is the DNA according to (2) or (3) or a DNA complementary to these DNAs and has a property of hybridizing to a Helicobacter pylori gene,
(9) For detection of Helicobacter pylori gene by polymerase chain reaction method (PCR method) containing at least two oligonucleotide primers selected from the group consisting of DNA according to (2) or (3) and DNA complementary thereto reagent,
(10) The reagent contains two oligonucleotide primers, which have the following properties:
(I) one primer hybridizes with a Helicobacter pylori gene chain, the other primer hybridizes with a gene chain complementary to the gene chain, and
(Ii) when the chain reaction product synthesized as a result of the amplification reaction is separated from its complement, it can function as a template for the synthesis of the chain reaction product of other primers;
The reagent according to (9), which has
About.
[0006]
The polypeptide of the present invention is not particularly limited as long as it has the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof, but more specifically, Preferred are those having the amino acid sequence or a part thereof and having reactivity with an anti-Helicobacter pylori antibody. In addition, as long as it has reactivity with anti-Helicobacter pylori antibody, the amino acid constituting the polypeptide or oligopeptide which is the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof Among the polypeptides of the present invention, so-called modified polypeptides or modified oligopeptides, such as polypeptides obtained by deleting one or more amino acids, substituting with other amino acids, or inserting other amino acids, are also included. included.
[0007]
The DNA of the present invention may be any DNA as long as it can encode the amino acid sequence represented by any of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof, but more specifically. Preferably, DNA having the base sequence represented by any of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof is used.
That is, a DNA having the amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof and a DNA encoding a polypeptide exhibiting a biological activity equivalent thereto Can be mentioned.
The DNA of the present invention may be obtained by any method. For example, it includes all of complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA obtained by chemical synthesis, and DNA constructed by appropriately combining these.
It goes without saying that the DNA of the present invention may be substituted with a corresponding RNA.
DNA encoding the polypeptide for the HP antigen of the present invention can be obtained by cloning cDNA from mRNA of the polypeptide, isolating from HP genomic DNA, chemically synthesizing, and the like.
For example, the following method is exemplified as a method for isolating DNA encoding a polypeptide derived from HP from HP genomic DNA.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
(1) First, HP is cultured in a liquid medium under microaerobic conditions, and HP cells are separated by centrifugation. The separated HP cells are preferably lysed with sodium dodecyl sulfate (SDS) and protease K, and the protein is denatured and removed by phenol / chloroform extraction, and then purified HP genomic DNA is prepared by ethanol precipitation [Slhavy, TJetal “Experiments with Gene Fusions” p.137-139 (1984)].
The obtained DNA is subdivided by partial digestion with an appropriate restriction enzyme or ultrasonic treatment or the like, and the resulting DNA fragment is incorporated into an appropriate phage vector, cosmid vector, plasmid vector or the like to construct a genomic DNA library.
The plasmid vector used here is not particularly limited as long as it can be replicated and maintained in the host, and any phage vector that can be propagated in the host may be used. However, in the case of subjecting to an immunological screening method described later, it is necessary to use a vector having a promoter capable of expressing an HP antigen in a host.
For example, Maniatis, T. et al., Molecular Cloning, A Laboratory Manual (Moleculer Cloning, A Laboratory Manual), cold Spring Harbor Laboratory, 1,82 (1982). And the like. As a method for incorporating DNA into a phage vector, the method of Hyunh, TV et al. [Hyunh, TV, DNA Cloning, Practical Approach (DNA Cloning.a practical approach) 1 , 49 (1985)]. The recombinant plasmid or phage vector thus obtained is introduced into a suitable host of prokaryotic cells or eukaryotic cells.
Examples of the method for isolating the DNA encoding the HP-derived polypeptide from the DNA library constructed by the above method include the following methods.
For example, after chemically synthesizing an oligonucleotide considered to correspond to a partial amino acid sequence of the HP-derived polypeptide, ³² Labeled with P as a probe, known colony hybridization method [Crunstein, M. and Hogness, DS: Proc. Natl. Acad. Sci. .USA) 72 : 3961 (1975)] or a plaque hybridization method (Moleculer Cloning, A Laboratory Manual, cold Spring Harbor Laboratory, 1,82 (1982)), clones containing the target DNA are selected. In addition, a method of selecting a clone having a target DNA by using an antigen-antibody reaction with an antibody against the HP-derived polypeptide (for example, an anti-CP2 antibody), or a specific region of the polypeptide gene by a polymerase chain reaction method (PCR method) ) To isolate the HP-derived polypeptide gene. If the entire region of the gene has not been obtained as a result of isolation, screening the genomic DNA library again by colony hybridization or plaque hybridization using the isolated DNA fragment or a part thereof as a probe Finally, the entire gene region can be obtained.
The base sequence of the DNA thus obtained was determined by the Maxam Gilbert method (Maxam, AMand Gilbert, W., Proc. Natl. Acad. Sci. USA, 74: 560 (1977)) or the dideoxynucleotide synthetic chain termination method. [Messing, J et al. Nucleic Acid Res 9 309 (1981)] and the presence of the HP-derived polypeptide gene can be confirmed. It can be obtained by cutting out all or part of the HP-derived polypeptide gene from the clone thus obtained with a restriction enzyme.
[0009]
(2) After chemically synthesizing an oligonucleotide considered to correspond to a partial amino acid sequence of a HP-derived polypeptide, ³² A probe labeled with P is prepared, and Southern blot hybridization is carried out with a restriction enzyme digestion product such as BamHI of the HP genome prepared in the same manner as in (1) above, thereby producing a restriction enzyme map near the target gene.
The prepared HP genomic DNA is digested with an appropriate restriction enzyme to fragment the DNA. The DNA piece is fractionated by a molecular weight fractionation method such as gel electrophoresis or gel filtration, and a fraction containing a DNA piece containing the target gene is collected with reference to the produced restriction enzyme map. A limited HP genomic DNA library is constructed by incorporating the sorted DNA fragments into a plasmid vector or phage vector.
A clone containing DNA encoding an HP-derived polypeptide is described above. ³² Selection is made by colony hybridization or plaque hybridization using a probe labeled with P. In addition, a method for selecting a clone having a target DNA using an antigen-antibody reaction with an antibody against the polypeptide (for example, an anti-CP2 antibody), or a specific region of the polypeptide gene by a polymerase chain reaction method (PCR method) And a method for isolating the polypeptide gene. If the entire region of the isolated polypeptide gene has not been obtained, Southern blot hybridization of HP genomic DNA is performed as described above using the isolated DNA fragment or a part of the DNA fragment as a probe. An HP genomic DNA fragment containing the remaining portion of the gene of interest is estimated from a group of DNA fragments obtained by various restriction enzyme digests. The HP genomic DNA library is constructed again with a group of DNA fragments that have been fractionated and sorted by the method described above, and a clone containing the target DNA is selected using the isolated DNA fragment or a part of the DNA fragment as a probe. By doing so, the entire gene region can be finally obtained. It can be obtained by cutting out all or part of the HP-derived polypeptide gene from the clone thus obtained with a restriction enzyme.
[0010]
Furthermore, the present invention is a recombinant vector containing DNA encoding the aforementioned HP-derived polypeptide or oligopeptide.
The recombinant vector is not particularly limited as long as it contains DNA encoding the above-mentioned HP-derived polypeptide or oligopeptide, and can be replicated and maintained in various prokaryotic and / or eukaryotic hosts. In other words, a recombinant vector constructed by a publicly known method (for example, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1, 82 (1982)).
As a vector used when constructing the recombinant vector of the present invention, a plasmid vector, a phage vector and the like are particularly suitable as long as they can be replicated and maintained in various prokaryotic and / or eukaryotic cells. Without limitation, natural plasmids, artificially modified plasmids (DNA fragments prepared from natural plasmids), synthetic plasmids and the like are used.
In addition, the recombinant vector of the present invention can also be easily prepared by incorporating DNA encoding the polypeptide or oligopeptide into a vector available in the art by a conventional method. Specific examples of such vectors include plasmids derived from E. coli such as pBR322, pBR325, pUC12, pUC13, and pBluescript, such as yeast-derived plasmids such as pSH19 and pSH15, and plasmids derived from Bacillus subtilis such as pUB110, pTP5 and pC194. Illustrated. Examples of phages include bacteriophages such as λ phage, and animal and insect viruses such as retroviruses, vaccinia viruses, and nuclear polyhedrosis viruses. Preferred are plasmid vectors and bacteriophage vectors.
In order to achieve the purpose of expressing DNA encoding HP-derived polypeptide or oligopeptide and producing these proteins, the DNA is incorporated into the expression vector when the recombinant vector of the present invention is constructed. Is desirable.
These expression vectors are those that can be replicated and maintained in various prokaryotic and / or eukaryotic hosts, and can detect HP in various prokaryotic and / or eukaryotic host cells. There is no particular limitation as long as it has a function of expressing protein DNA, in other words, a function of producing a target antigen protein.
Such expression vectors available in the art include E. coli host cells. In the case of E. coli, for example, pBR322, pUC12, pUC13, pTrcHis, pMAL-c2, pMAL-p2, or an artificially modified product thereof (a DNA fragment obtained by treating the vector with an appropriate restriction enzyme), etc. Is yeast, for example, plasmids pRS403, pRS404, pRS413, pRS414, pYES2, etc., and when the host cell is an animal cell, for example, plasmid pRSVneo ATCC 37224, pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37, etc. However, when the host cell is an insect cell, for example, Autographica californica nuclear polyhedrosis virus (AcNPV), Bombyx mori Nuclear polyhedrosis virus (BmNPV) and the like are preferably mentioned.
Bacteria as host cells, particularly E. coli. When using E. coli, the recombinant vector of the present invention generally comprises a promoter-operator region, a start codon, DNA encoding the polypeptide or oligopeptide of the present invention, a stop codon, a terminator region, and the like. is there.
When yeast or animal cells are used as host cells, the recombinant vector of the present invention generally comprises a promoter, a start codon, DNA encoding the HP-derived polypeptide or oligopeptide of the present invention, a stop codon, etc. Is. In addition, it is optional to incorporate the DNA encoding the signal peptide, the enhancer sequence, the 5′-side and 3′-side untranslated regions of the polypeptide or oligopeptide derived from the present invention into the recombinant vector.
[0011]
The promoter-operator region for expressing the HP-derived polypeptide of the present invention in bacteria contains a promoter, an operator, and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is a genus Escherichia, those preferably containing Trc (trp-lac) promoter, Tac promoter, Trp promoter, lac promoter, recA promoter, λPL promoter, 1pp promoter, etc. are exemplified. Examples of promoters for expressing the polypeptide of the present invention in yeast include PHO5 promoter, PGK promoter, GAP promoter, and ADH promoter. When the host is Bacillus, SLO1 promoter, SP02 promoter, penP promoter, etc. Can be mentioned. When the host species is a eukaryotic cell such as an animal cell, examples include SV40-derived promoters, retrovirus promoters, and nuclear polyhedrosis virus promoters. However, it is not particularly limited to these.
In addition, for expression, a method of inducing expression by adding isopropyl-β-D-thiogalactoside (IPTG) and use of an enhancer are also effective methods.
A suitable initiation codon is exemplified by methionine codon (ATG).
Examples of the stop codon include common stop codons (for example, TAG, TGA, etc.).
The terminator region includes a natural or synthetic terminator. Examples of the enhancer sequence include SV40 enhancer sequence (72 bp), DNA tumor viruses such as polyoma, adeno, papilloma, retrovirus LTR (Long Terminal Repeat), immunoglobulin H chain, L chain gene, and the like. An expression vector can be prepared by linking a promoter, an initiation codon, DNA encoding the HP-derived polypeptide of the present invention, a termination codon and a terminator region continuously and circularly to appropriate replicable units. At this time, an appropriate DNA fragment (for example, a linker or the like) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase, if desired.
[0012]
The transformant of the present invention (hereinafter used in a concept including a transfectant) can be prepared by introducing the above-described expression vector into a host cell.
Examples of host cells include microorganisms [bacteria (for example, Escherichia, Bacillus), yeast (for example, Saccharomyces), animal cells, insect cells, and the like). Specifically, in the genus Escherichia, Escherichia coli DH1, M103, JA221, HB101, C600, XL-1 Blue, JM109, TOP10 and the like are exemplified. Examples of Bacillus include Bacillus subtilis MI114, 207-21. Saccharomyces cerevisiae AH22, AH22R as yeast ^- NA87-11A, DKD-5D, etc. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO, mouse L cells, and human L cells. Examples of insect cells include BmN4 and Sf9, but are not particularly limited thereto.
Prokaryotic cells are generally preferred as host cells for DNA sequence cloning and vector assembly. The assembled vector is then transformed into appropriate host cells, in which case prokaryotic and eukaryotic cells can be used.
Introduction of an expression vector into a host cell [transformation (including transfection)] can be performed using a conventionally known method.
In the case of bacteria (for example, E. coli and Bacillus subutilis), for example, the method of Cohen et al. [Proc. Natl. Acad. Sci. USA, (1972) 69, 2110], the protoplast method [molecular gene of genetics (Mo1.Gene.Genet.), (1979) 168 , 111] or competent method [J. Mol. Biol., (1971) 56 In the case of Saccharomyces cerevisiae, for example, the method of Hinnnen et al. [Proc. Natl. Acad. Sci. USA, (1978) 75, 1927] or the lithium method [Journal of Bacteriol. ), (1983) 153 In the case of animal cells, for example, Graham's method [Virology, (1973) 52 , 456] etc., but is not particularly limited thereto.
[0013]
The HP-derived polypeptide of the present invention can be produced by culturing a transformant containing the expression vector prepared as described above in a nutrient medium.
The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large Examples include soybean cake and potato extract. If desired, other nutrients [for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, ampicillin, kanamycin, etc.), etc.] may be included.
Culturing is performed according to methods known in the art. The culture conditions such as temperature, medium pH and fermentation time are appropriately selected so that the highest titer of the polypeptide as an antigen can be obtained.
In addition, although the specific culture medium and culture | cultivation conditions used according to a host are illustrated below, it is not limited to this at all.
When the host is a bacterium, actinomycetes, yeast, or filamentous fungus, for example, a liquid medium containing the above nutrient source is suitable. At that time, the pH is preferably 5-8.
When the host is E. coli, LB medium, YT medium, SOB medium [Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1, 82 (1982)], M9 medium [Miller. Journal of Experimental Molecular Genetics (J. Exp. Mol. Genet.), (1972) p. 431, Cold Spring Harbor Laboratory, New York]. In such a case, the culture can be performed usually at 14 to 42 ° C., preferably 28 to 39 ° C. for about 3 to 24 hours, with aeration and stirring as necessary.
When the host is a Bacillus genus, the treatment can be performed usually at 14 to 42 ° C., preferably at 28 to 39 ° C. for about 3 to 96 hours, with aeration and stirring as necessary.
When the host is yeast, examples of the medium include a medium developed by Bastian et al. [Bostian.KLet.al (1980) Proc. Natl. Acad. Sci. USA, 77, 4505], and the pH is 5-8. It is desirable that The culture is usually performed at 14 to 42 ° C., preferably 20 to 35 ° C. for about 12 hours to 10 days, and if necessary, aeration or agitation can be performed.
When the host is an animal cell, the MEM medium containing about 5 to 20% fetal bovine serum as a medium [Science (1952) 122 , 501], DMEM medium [Virology, (1959) 8 , 396], RPMI 1640 medium [J. Am. Med. Assoc., (1967) 199 , 519], 199 medium [Proc. Soc. Exp. Biol. Med., (1950), Proceedings of Society of Experiments of Biological Medicine (1950) 73 , 1] etc. can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to 40 ° C., preferably at 34 to 38 ° C. for about 12 to 72 hours, and aeration and agitation can be performed as necessary.
When the host is an insect cell, for example, Grace's medium containing fetal calf serum (Proc. Natl. Acad. Sci. USA (1985) 82 , 8404], etc., and the pH is preferably about 5-8. The culture is usually carried out at about 20 to 40 ° C., preferably 25 to 30 ° C. for about 12 hours to 10 days, and aeration and agitation can be performed as necessary.
[0014]
The HP-derived polypeptide of the present invention can be obtained from the culture obtained by the above culture as follows.
That is, when the HP-derived polypeptide of the present invention is present in a culture solution among the cultures, a culture filtrate (supernatant) is obtained from the obtained culture by a method such as filtration or centrifugation, and the culture solution is obtained. The polypeptide is purified and isolated according to conventional methods commonly used to purify and isolate natural or synthetic proteins from
Examples of isolation and purification methods include molecular weights such as methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method, etc. Method using specific difference, method using specific affinity such as affinity chromatography, method using hydrophobic difference such as reversed phase high performance liquid chromatography, isoelectric point difference such as isoelectric focusing The method of using is mentioned.
On the other hand, when the HP-derived polypeptide of the present invention is present in the periplasm or cytoplasm of a cultured transformant, the culture is collected according to a conventional method such as centrifugation, and the cells are collected in an appropriate buffer. After suspending and disrupting cell walls and / or cell membranes such as cells by a method such as ultrasonic wave, lysozyme and freeze-thawing, a crudely purified fraction containing the polypeptide is obtained by a method such as centrifugation or filtration. The crudely purified fraction can be isolated and purified according to the conventional method shown above.
[0015]
The polypeptide of the present invention can be used as an antigen in immunoassays such as enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescent immunoassay (FIA) known per se. Detection of anti-HP antibodies in them can be performed.
More specifically, for example, first, the polypeptide immobilized on a suitable carrier is reacted with a sample to form the polypeptide-anti-HP antibody complex, and this is reacted with a labeled anti-human immunoglobulin antibody. After that, the anti-HP antibody is measured using the label, or the polypeptide is labeled, then reacted with the sample, B / F separated by a conventional method, and then the labeled product is used for the anti-HP antibody. The method etc. which measure are mentioned. The polypeptide used in the immunological assay may be a part of the polypeptide as long as it can be used as an antigen against the anti-HP antibody, or a part of the polypeptide obtained by chemical synthesis or the like. It may be a chain. Further, it may be a polypeptide such as a fusion protein containing the polypeptide, a part of the polypeptide and the peptide chain.
As described above, these peptides can be used as reagents for measuring anti-HP antibodies. For example, a polypeptide as described above, a part of the polypeptide, the peptide chain, or a polypeptide containing them is fixed, for example, a microplate, When an enzyme immunoassay is performed using a carrier such as a bead, an enzyme-labeled anti-human immunoglobulin antibody, and an anti-HP antibody measurement kit containing a substrate that develops color or luminescence or fluorescence, HP in the sample is specifically and highly enriched. Sensitivity can be detected.
Furthermore, since these peptides have reactivity with anti-HP antibodies, that is, have antigenic determinants recognized by anti-HP antibodies, they produce anti-HP antibodies in vivo. (The ability to increase specific immunity against HP). Therefore, the peptide of the present invention can be used as a vaccine against so-called HP, which prevents HP infection and onset after infection.
In addition, if oligonucleotide primers for PCR are appropriately prepared based on the base sequence of the DNA of the present invention and DNA complementary thereto, for example, PCR using gastric juice or pathological tissue such as stomach or duodenum as a sample It is also possible to detect HP in a sample specifically and with high sensitivity by the method. Furthermore, similarly, if an oligonucleotide or polynucleotide is appropriately prepared based on the base sequence of the DNA of the present invention or a DNA complementary thereto, and this is used as a labeled probe, the HP in the sample can be obtained. Can be detected specifically and with high sensitivity.
Thus, these DNAs can be used as HP detection reagents and contain, for example, oligonucleotide primers appropriately selected as described above, thermostable DNA polymerase and its substrate dNTP (dATP, dCTP, dGTP, dTTP). If a PCR method or a ligase chain reaction (LCR) method is performed using an HP gene detection kit, HP in a sample can be detected specifically and with high sensitivity.
Currently known PCR methods include PCR-SSCP (PCR single-strand conformation polymorphism) method, RT-PCR (reverse transcription PCR) method, LA-PCR (long and accurate PCR) method, inverse-PCR method, SSP- There are PCR (single specific PCR) methods, etc., and these are related to Yodosha “Bio-manual UP series, latest technology of PCR method”, edited by Kenji Hayashi, etc., February 5, 1995, first print, October 25, 1995. Please refer to the 2nd printing issue. Regarding the LCR method, please refer to Tokyo Chemical Doujin, “Contemporary Encyclopedia, Biotechnology (2nd edition)” Jun Marunouchi et al., Issued on June 1, 1994.
The reagent by the PCR method using the oligonucleotide primer contains two oligonucleotide primers, and the oligonucleotide primer has the following properties:
(I) one primer hybridizes with a Helicobacter pylori gene chain, the other primer hybridizes with a gene chain complementary to the gene chain, and
(Ii) when the chain reaction product synthesized as a result of the amplification reaction is separated from its complement, it can function as a template for the synthesis of the chain reaction product of other primers;
It is preferable that it has.
Examples of the primers for performing various PCRs as described above include the following sequences.
In particular, those showing a common sequence (homology) among HP strains are effective in detecting the HP gene.

Alternatively, for example, if a hybridization method is performed using an HP gene detection kit containing a polynucleotide probe or oligonucleotide probe appropriately selected as described above, HP in the sample can be detected specifically and with high sensitivity. . The HP gene includes RNA in addition to DNA.
In the following Examples, plasmids, for example, enzymes such as restriction enzymes and T4 ligase, and other substances were commercially available, and the usage methods were in accordance with ordinary methods. In addition, procedures for cloning DNA, transforming host cells, culturing transformants, and collecting and purifying HP-specific detection polypeptide antigens from the resulting culture are well known in the art. It is a method that can be known from things or documents.
Reference Examples and Examples will be given below, but the present invention is not limited to these descriptions.
[0016]
【Example】
[Example 1] Determination of base sequence of DNA encoding CP2 antigen and amino acid sequence of CP2 antigen
(1) Determination of N-terminal amino acid sequence of CP2 antigen
HP (ATCC 43504; Rockville, MD) was inoculated on a Brucella (Difco Laboratories, Detroit, MD) agar plate containing 10% horse blood and cultured at 37 ° C for 5 days under microaerobic conditions. The grown HP colonies were inoculated into 450 ml of Brucella medium containing 10% horse serum and cultured at 37 ° C. for 5 days under microaerobic conditions. The cells were collected by centrifugation (6000 rpm) and washed with PBS (0.1 M Sodium-phosphate, pH 7.4, 0.15 M NaCl). The cells are disrupted and solubilized with ultrasound, and a fraction of about 60 KD of SDS-PAGE [Anti-CP2 monoclonal antibody (Sugiyama, T et al. , Gastroenterology 101, 77-83, 1991). This was repeated three times, and then introduced into a Sepharose 4B column on which the CP2 monoclonal antibody was immobilized. The protein fraction adsorbed on the column was eluted with 0.2 M glycine hydrochloride buffer to obtain a purified CP2 antigen fraction. This was further purified by SDS-PAGE using a prep cell, and the electrophoretically single CP2 antigen was used for amino acid sequence analysis.
The purified CP2 antigen was subjected to an automatic protein sequencer (ABI, 477-A type) and the N-terminal amino acid sequence was examined. As a result, the following 20 amino acid sequences were revealed.
Met Val Asn Lys Asp Val Lys Gln Thr Thr Ala Phe Gly Ala Pro Val Trp AspAsp Asn
This sequence was in complete agreement with the sequence reported as the N-terminal sequence of HP catalase, Met Val Asn Lys Asp Val Lys Gln Thr Thr Ala Phe Gly Ala Pro Val (Westblom, TU et al, Eur. J. Clin. Microbiol. Infect. Dis., Vol11. No.6, 522-526, June 1992).
[0017]
(2) Preparation of HP genomic DNA
About 0.5 g (wet weight) of HP cells (ATCC43504) was washed twice with 10 ml of ice-cooled PBS (0.1 M sodium-phosphate, pH 7.4, 0.15 M NaCl) by centrifugation, and then 2.5 ml Of 50 mM Tris-HCl (pH 8.0). 120 μl of 10% SDS and 0.5 ml of STEP (0.5% SDS, 50 mM Tris-HCl (pH 7.5), 0.4 M EDTA 1 mg / ml protease K) were added thereto and reacted at 50 ° C. for 2 hours. After the reaction, 2 ml of TE buffer (pH 8.0) was added and extracted twice with 5 ml of phenol / chloroform to remove the protein. Furthermore, after extracting twice with an equal amount of ether, 2.5 times the amount of ethanol was added, and the DNA and RNA mixture was collected by precipitation by centrifugation. The precipitate was dried, dissolved in 1 ml of TE buffer, 200 μg of RNase A was added, and the mixture was kept at 37 ° C. for 1 hour. After extraction twice with an equal amount of phenol / chloroform, extraction with ether and ethanol precipitation yielded approximately 1 mg of purified DNA.
(3) Cloning of CP2 antigen gene
An oligonucleotide (17 bases) synthesized from the sequence information of the N-terminus of CP2 antigen ³² Southern blot hybridization analysis of digested products of various restriction enzymes of HP genomic DNA (PstI, HindIII, StuI, HaeIII, Sau3AI, SacI, XbaI, BamHI, ApaI, ClaI, XhoI, etc.) went. As a result, it was revealed that the target DNA that reacts with the probe becomes a fragment of about 400 bases by digesting HP genomic DNA with the restriction enzyme Sau3AI.
200 μg of HP genomic DNA was digested with Sau3AI at 37 ° C. for 16 hours, the digested product was subjected to agarose gel electrophoresis, and the gel at the position where the DNA fragment of about 400 bases that reacts with the probe was migrated was separated. DNA was eluted from the separated gel by electrophoresis and purified by phenol / chloroform extraction. The purified DNA was recovered by ethanol precipitation, and the precipitate was washed with 70% ethanol and dried to obtain a DNA fragment group of about 400 bases. This was inserted into the BamHI site of pBLuescriptIISK + to transform E. coli (JM109), and thousands of transformant colonies were obtained on an agar plate.
The colonies on the agar plate were transferred to a nitrocellulose membrane and immersed in an alkaline denaturing solution (1.5M NaCl, 0.5M NaOH) for 5 minutes for lysis and DNA denaturation. Next, the nitrocellulose membrane was immersed in a neutralization solution (1M Tris-HCl pH 8.0, 1.5M NaCl) for 5 minutes, immersed in 2 × SSC (0.3M NaCl, 0.03M sodium citrate) for 5 minutes, and then at 80 ° C. For 2 hours to fix the DNA on the membrane. The membrane was immersed in a hybridization solution [6 × SSC, 0.1% SDS, 20 mM Tris-HCl (pH 8.0), 0.125% sodium caseinate, 2.5 mM EDTA, 100 μg / ml denatured salmon sperm DNA] at 42 ° C. for 1 hour. After, γ ³² The above-mentioned oligonucleotide (17 bases) labeled with P-ATP was added and reacted at a temperature gradient of −4 ° C. per hour from 50 ° C. to 30 ° C., followed by further reaction at 30 ° C. for 8 hours. The nitrocellulose membrane was removed from the solution, washed with 2 × SSC for 15 minutes and twice at room temperature, and then autoradiographed.
Colonies determined to be positive by autoradiography were purified to obtain a plasmid pSKC5 into which a 440-base DNA containing the N-terminal gene of the CP2 antigen was inserted.
By analogy with the restriction enzyme map obtained from this result and the Southern blot hybridization analysis described above, it is considered that the C-terminal gene of the CP2 antigen is contained in a DNA fragment of about 600 bp fragmented by PstI and Sau3AI. It was. Therefore, in order to obtain the remaining region, cloning experiments were carried out as follows.
200 μg of HP genomic DNA was partially digested with HaeIII, and the digestion product was subjected to agarose gel electrophoresis to separate a fraction of DNA fragments of 4.5 Kbp to 3 Kbp that reacted with the probe. Further, this fraction was digested with PstI at 37 ° C. for 3 hours, and the digested product was subjected to agarose gel electrophoresis to separate a DNA fragment of about 1.8 Kbp which reacts with the probe. The separated DNA was digested with Sau3AI at 37 ° C. for 1 hour, and the digested product was cloned into a plasmid vector (pMAL-c2) having a PstI-BamHI cleavage surface. As a result, plasmid pMBP26 was obtained in which about 600 bp of DNA fragment containing the gene on the C-terminal side of CP2 antigen was inserted.
The base sequence of the CP2 antigen gene contained in pSKC5 and pMBP26 was determined by the dideoxy method using DNA Taq Polymerase. PCR using two synthetic oligonucleotides AAGATGGTTAATAAAGATGTG (18th to 38th base of SEQ ID NO: 1) and AAAATCAATGCTGTATTGAGC (reverse strand of 1800 to 1820th base of SEQ ID NO: 1) prepared with reference to this sequence as primers The fragment containing the CP2 antigen structural gene was amplified by performing the method (94 ° C., 300 seconds: 94 ° C., 90 seconds, 60 ° C., 120 seconds, 72 ° C., 180 seconds, 30 cycles: 72 ° C., 120 seconds) . The resulting amplified fragment was blunted at both ends, added with a phosphate group, and cloned into a pTrcHis expression vector (manufactured by Invitrogen) having a blunted EcoRI cut surface, and a plasmid containing the CP2 antigen structural gene pTrcCP2 was obtained.
From the resulting plasmid pTrcCP2, the base sequence of the inserted fragment (CP2 fragment) was determined by the dideoxy method using DNA Taq Polymerase.
As a result of the analysis of the sequence, an HP genomic DNA sequence 1829 bp (SEQ ID NO: 1) containing the sequence 1515 bp of the CP2 antigen structural gene was revealed. As a result, the amino acid sequence of the CP2 antigen (SEQ ID NO: 1) consisting of 505 amino acid residues having ATG starting from the 21st base counting from the 5 ′ end as the translation initiation sequence was clarified.
When the homology search of the result of this arrangement | sequence was carried out with EMBL of Genetics gene analysis software (SDC software development company), and the data bank of GenBank, 50% or more homology was shown with catalases, such as a rat and a human.
[0018]
[Example 2] Expression of recombinant CP2 antigen protein
The resulting expression vector pTrcCP2 transformed into E. coli (JM109) is cultured overnight in LB medium, and then 20 μl of the culture is inoculated into about 3 ml of LB medium, resulting in an OD600 of 0.6. Until cultured. Two of them were prepared, and one of them was added with IPTG to a final concentration of 1 mM, and further cultured for 3 hours. After centrifugation (5000rpm, 10min) for 2 samples after culturing, double volume of SDS-PAGE loading Buffer [125mM Tris-HCl (pH6.8), 4% SDS, 0.2% BPB, 20% glycerol] Was added and boiled for 3 minutes, and SDS-PAGE was performed. As a result, recombinant CP2 antigen protein induced and expressed by IPTG could be confirmed.
[0019]
[Example 3] Reaction of recombinant CP2 antigen protein with anti-CP2 monoclonal antibody (Sugiyama, T et al, Gastroenterology 101, 77-83, 1991)
After completion of SDS-PAGE in Example 2, the protein in the polyacrylamide gel was further transferred to a nitrocellulose membrane using a semi-drive lotter (Sartorius). Next, the nitrocellulose membrane was shaken in a blocking solution (1% BSA, 20 mM Tris-HCl pH 7.5, 150 mM NaCl) for 1 hour, and then anti-CP2 monoclonal antibody (derived from mouse) was added and shaken for another hour. The nitrocellulose membrane was removed from the solution, washed with TBST [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20 (V / V)] three times for 5 minutes, and then TBS (20 mM Tris-HCl pH 7.5, 150 mM). NaCl) was further washed 5 times for 5 minutes. Next, the nitrocellulose membrane is immersed in an alkaline phosphatase-labeled anti-mouse IgG antibody solution diluted with a blocking solution for 1 hour, then removed from the solution, washed 3 times with TBST for 5 minutes, and further washed 5 times with TBS for 10 minutes. did. After washing, the nitrocellulose membrane was subjected to coloring solution (0.3 mg / ml Nitro Blue Tetrazolium, 0.15 mg / ml 5-Bromo-4-Chloro-3-Indolil Phosphate, 100 mM Tris-HCl pH 9.5, 100 mM NaCl, 5 mM MgCl ₂ ) Color was developed.
As a result, it was confirmed that the recombinant CP2 antigen protein reacts with the anti-CP2 monoclonal antibody.
[0020]
[Example 4] Acquisition of CP2 antigen gene having different primary structure
(1) Acquisition of CP2 antigen gene from HP cells (ATCC43504)
The HP genomic DNA prepared from the HP cells (ATCC 43504) in Example 1 (1) was synthesized based on the HP genomic sequence (SEQ ID NO: 1) containing the CP2 antigen structural gene determined in Example 1. PCR method (94 ° C., 300 seconds: 94 ° C.) using primers of various types of oligonucleotides AAGATGGTTAATAAAGATGTG (18th to 38th base of SEQ ID NO: 1) and AAAATCAATGCTGTATTGAGC (reverse strand of 1800 to 1820th base of SEQ ID NO: 1) 90 seconds, 60 ° C., 120 seconds, 72 ° C., 180 seconds, 30 cycles: 72 ° C., 120 seconds), the fragment containing the CP2 antigen structural gene was amplified. After blunting both ends of the obtained amplified fragment and adding a phosphate group, it was cloned into a pBluescript vector having a SmaI cut surface and a pTrcHis expression vector having a blunted EcoRI cut surface, and CP2 Plasmid pBluePCR1 and plasmid pHisPCR1 containing the antigen structural gene were obtained.
From the resulting plasmid pBluePCR1, various mutant mutation lengths were prepared using a dilation kit (Nippon Gene), and an insert fragment (CP2) was prepared by the dideoxy method using an ALFred AutoRead Seqencing kit (Pharmacia). The base sequence of -PCR1 fragment) was determined.
As a result of analysis of the sequence, an HP genome sequence 1803 bp (SEQ ID NO: 2) containing the sequence 1515 bp of the CP2 antigen structural gene was revealed. As a result, the CP2 antigen structural gene in this CP2-PCR1 fragment differs from the CP2 antigen structural gene obtained in Example 1 in two base sequences and in two amino acid sequences when translated. It became clear that it was a thing.
The homology comparison result between the amino acid sequence of the CP2 antigen structural gene in the CP2-PCR1 fragment and the amino acid sequence of the CP2 antigen structural gene obtained in Example 1 is shown in FIG.
(2) Acquisition of CP2 antigen gene from HP clinical strain
HP genomic DNA was prepared from HP cells collected from the stomach of a chronic gastritis patient (24 years old, male) in the same manner as in Example 1 (1).
The obtained HP genomic DNA was subjected to the PCR method (94 ° C., 300 seconds: 94 ° C., 90 seconds, 60 ° C., 120 seconds) using two kinds of synthetic oligonucleotides similar to (1) above as primers. , 72 ° C., 180 seconds, 45 cycles: 72 ° C., 120 seconds), and a fragment containing the CP2 antigen structural gene was amplified. Both ends of the obtained amplified fragment were blunted, added with a phosphate group, and then cloned into a pBluescript vector having a SmaI cut surface to obtain a plasmid pBluePCR2 containing a CP2 antigen structural gene. Further, from the obtained plasmid pBluePCR2, an insert fragment (CP2-PCR2 fragment) was cut out with BamHI and XhoI, cloned into a pTrcHis expression vector having a BamHI / XhoI cut surface, and a plasmid pHisPCR2 containing a CP2 antigen structural gene was obtained. Obtained.
From the obtained plasmid pBluePCR2, as in the above (1), a mutation mutant with various base lengths was prepared using a delation kit (manufactured by Nippon Gene), and an ALFred AutoRead Seqencing kit (manufactured by Pharmacia) was used. The base sequence of the inserted fragment (CP2-PCR2 fragment) was determined by the dideoxy method.
As a result of analysis of the sequence, an HP genome sequence 1810 bp (SEQ ID NO: 3) containing the sequence 1515 bp of the CP2 antigen structural gene was revealed. As a result, the CP2 antigen structural gene in this CP2-PCR2 fragment differs from the CP2 antigen structural gene obtained in Example 1 in 63 nucleotide sequences and 15 amino acid sequences when translated. It became clear that it was a thing.
The homology comparison result between the amino acid sequence of the CP2 antigen structural gene in the CP2-PCR2 fragment and the amino acid sequence of the CP2 antigen structural gene obtained in Example 1 is shown in FIG.
In FIG. 1, G is glycine, A is alanine, V is valine, L is leucine, I is isoleucine, S is serine, T is threonine, D is aspartic acid, E is glutamic acid. N is asparagine, Q is glutamine, K is lysine, R is arginine, C is cysteine, M is methionine, F is phenylalanine, Y is tyrosine, W is tryptophan, H is histidine And P represents proline, respectively.
In FIG. 1, the upper CP2 shows the amino acid sequence of the CP2 antigen structural gene obtained in Example 1, and the middle CP2-PCR1 shows the CP2 antigen structural gene (CP2-antigen) obtained in Example 4 (1). The amino acid sequence of PCR1) and the lower CP2-PCR2 show the amino acid sequence of the CP2 antigen structural gene (CP2-PCR2) obtained in Example 4 (2), respectively.
Furthermore, the * mark in FIG. 1 indicates that the amino acid sequence of the CP2 antigen structural gene is an amino acid in which a difference is recognized among the three.
From the results of FIG. 1, it is found that the HP-derived CP2 antigen structural gene has small mutations within the same strain and large mutations due to differences in strains. In other words, there are several HP-derived CP2 antigens. It can be seen that a mutant type exists.
In addition, this result is also reported with Westblom, TU et al. Report of HP mutant strains showing negative catalase activity (Eur. J. Clin. Microbiol. Infect. Dis., Vol 11, No. 6, 522-526, June 1992). Match.
[0021]
[Example 5] Expression of recombinant CP2 antigen protein and reaction with anti-CP2 monoclonal antibody
The two types of recombinant vectors pHisPCR1 and pHisPCR2 obtained in Example 4 were transformed into E. coli (TOP10), respectively, and cultured in the same manner as in Example 2. After completion of the culture, centrifugation was performed in the same manner as in Example 2, and SDS-PAGE was performed in the same manner as in Example 2 to confirm the presence or absence of expression of the recombinant CP2 antigen protein. Furthermore, the reactivity between the recombinant CP2 antigen protein and the anti-CP2 monoclonal antibody was confirmed by the same Western blotting method as in Example 3.
The results of SDS-PAGE of CP2-PCR1 recombinant protein are shown in FIG. 2 (a), and the results of Western blotting analysis with the anti-CP2 monoclonal antibody using the SDS-PAGE are shown in FIG. 2 (b). .
The results of SDS-PAGE of the CP2-PCR2 recombinant protein are shown in FIG. 3 (a), and the results of Western blotting analysis with the anti-CP2 monoclonal antibody using the SDS-PAGE are shown in FIG. 3 (b). Each one is shown.
In addition, each lane number in a figure shows the result of using the following samples, respectively.

2 (a) and 3 (a) indicate the position of the recombinant CP2 antigen protein, and the → marks in FIGS. 2 (b) and 3 (b) indicate the recombination. The position of the reaction signal between the CP2 antigen protein and the anti-CP2 monoclonal antibody is shown.
As is clear from the results of FIGS. 2 (a) and 3 (a), transformants containing the recombinant vectors pHisPCR1 and pHisPCR2 are both about 60 KD recombinant CP2 antigen protein (CP2-PCR1 pair) by IPTG. It can be seen that the recombinant protein and the CP2-PCR2 recombinant protein) are efficiently induced and expressed.
The recombinant protein is expressed as a fusion protein containing a hexamer of histidine residues, and therefore has a slightly higher molecular weight than the CP2 antigen in the HP-producing protein.
As is clear from the results of FIGS. 2 (b) and 3 (b), both CP2-PCR1 recombinant protein and CP2-PCR2 recombinant protein are antibodies against HP-derived CP2 antigen, ie, anti-CP2 It can be seen that it has reactivity with the monoclonal antibody.
Further, a protein having a molecular weight smaller than that of the recombinant CP2 antigen protein (about 60 KD) of the present invention is observed. This is a protein obtained by cleaving the recombinant CP2 antigen protein due to degradation in the host cell. it is conceivable that. Since these recombinant proteins also have reactivity with the anti-CP2 monoclonal antibody, the partial sequence of the sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the present invention It was also suggested that it can be used as an antigen for HP-specific detection.
[0022]
[Example 6] Reaction of recombinant CP2 antigen protein with patient serum
Each of the two recombinant vectors pHisPCR1 and pHisPCR2 obtained in Example 4 was transformed into E. coli (TOP10), and 50 ml of SOB medium (2% tryptophan, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO _Four ) OD ₆₀₀ Each of the cells was cultured until it reached 0.6, and further IPTG was added to a final concentration of 1 mM, followed by culturing for 2 to 5 hours to induce expression by IPTG. After completion of the culture, the cells were collected by centrifugation (5000 rpm, 10 min) and suspended in 10 ml of lysis buffer (6 M Guanidium Hydrochloride, 20 mM Sodium phosphate, 500 mM Sodium chloride). The suspension obtained was sonicated to disrupt the cells and then centrifuged (3000 G, 15 min) to remove the precipitate. The resulting supernatant was treated with a ProBond resin column (Invitrogen: nickel column) And CP2-PCR1 and CP2-PCR2 recombinant CP2 antigen protein were affinity purified (recombinant CP2 antigen protein is expressed as a fusion protein having a hexamer of histidine residues, and thus has affinity for nickel. Is shown.) The obtained recombinant CP2 antigen protein was subjected to SDS-PAGE in the same manner as in Example 2, and then the protein in the polyacrylamide gel was transferred to a nitrocellulose membrane using a semi-drive lotter (Sartorius). Next, the nitrocellulose membrane was placed in a 3 ml blocking solution [25% block Ace (Dainippon Pharmaceutical Co., Ltd., TBS buffer containing 1% BSA)] for 1 hour at room temperature, and then the duodenal ulcer patient (HP infected person) 5 μl of serum was added and allowed to react for another hour. After the reaction, the nitrocellulose membrane was taken out and washed with TBST buffer, and then reacted for 1 hour in a blocking solution to which an alkaline phosphatase-labeled anti-human IgG antibody (manufactured by Kappel) was added. As a control, Western blotting analysis was performed in the same manner using the serum of a healthy person (HP non-infected person) instead of the patient serum.
The results of SDS-PAGE of CP2-PCR1 recombinant protein and CP2-PCR2 recombinant protein are shown in FIG. 4 (a), and the results of Western blotting with sera of duodenal ulcer patients using the SDS-PAGE are shown. Each is shown in 4 (b).
The results of SDS-PAGE of CP2-PCR1 recombinant protein and CP2-PCR2 recombinant protein are shown in FIG. 5 (a), and Western blotting analysis with normal (healthy person) serum using the SDS-PAGE. The results are shown in FIG.
In addition, each lane number in a figure shows the result of using the following samples, respectively.

4 (a) and FIG. 5 (a) indicate the position of the recombinant CP2 antigen protein, and the → mark in FIG. 4 (b) indicates the recombinant CP2 antigen protein and serum. The position of the reaction signal with the antibody against HP in FIG. 5 (b) indicates the position where the reaction signal between the recombinant CP2 antigen protein and the antibody against HP in serum will occur.
As is apparent from the results of FIGS. 4 (a) and 5 (a), about 60 KD of purified recombinant CP2 antigen protein (purified CP2-PCR1 recombination) was obtained from transformant cultures containing the expression vectors pHisPCR1 and pHisPCR2, respectively. It can be seen that protein and purified CP2-PCR2 recombinant protein) are obtained. The recombinant protein is expressed as a fusion protein containing a hexamer of histidine residues. Therefore, the recombinant protein is slightly more in comparison with CP2 antigen in HP-producing protein and CP2 antigen purified from HP cells. High molecular weight.
As is clear from the results of FIG. 4 (b), the purified recombinant CP2 antigen protein thus obtained is an anti-CP2 antibody that reacts with the CP2 antigen in the HP-producing protein and the CP2 antigen purified from the HP cells. It can be seen that it reacts with anti-CP2 antibody in the serum of patients with duodenal ulcer (HP infected). Further, as is apparent from the results of FIG. 5 (b), it can be seen that the obtained purified recombinant CP2 antigen protein does not react with components in normal (healthy) serum.
From the above, if the recombinant CP2 antigen protein of the present invention is used, it is possible to detect an antibody against HP present in the serum of a patient (HP infected person), that is, whether serum is infected or not. When the transformant of the present invention is cultured and the recombinant CP2 antigen protein is separated and purified from the culture, a polypeptide useful as an antigen for specifically detecting HP can be easily obtained. It can be seen that it can be obtained in large quantities.
[0023]
[Example 7] Evaluation of recombinant CP2 antigen by sandwich ELISA
100 μl of 10 μg / ml concentration of anti-CP2 monoclonal antibody was dispensed into a 96-well plate, and allowed to stand at 4 ° C. overnight to immobilize the antibody. The plate was washed three times with PBS (10 mM sodium phosphate, 0.15 M sodium chloride) after solid-phase treatment.
Next, 400 μl of blocking solution (PBS, 25% blockAce) was dispensed into the wells of the plate and left to stand at room temperature for 6 hours for blocking treatment, followed by washing with PBS three times.
After dispensing 100 μl of a recombinant CP2 antigen (CP2-PCR1, CP2-PCR2 obtained in Example 6) solution at a concentration of 10 μg / ml into the wells of the plate subjected to blocking treatment, the plate was allowed to stand at 4 ° C. overnight. Washed 3 times with PBS.
Next, dispense 100 μl each of serum from patients with gastric ulcer, gastric cancer, duodenal ulcer, etc. (10 samples) or normal (healthy) serum (5 samples) diluted 1000-fold with blocking solution and leave at 4 ° C overnight. And then washed 5 times with PBS.
Dispense 100 μl of coloring solution [10 mg o-phenylenediamine, 10 μl 30% hydrogen peroxide, 10 ml phosphoric acid citrate buffer (0.1 M citric acid, 0.2 M sodium hydrogen phosphate, pH 5.0)] After 5 minutes of reaction at room temperature, the reaction was stopped with 100 μl of 1N sulfuric acid.
The absorbance of each well was measured with a Tmax plate reader (manufactured by Molecular Devices) (measure: 490 nm, reference: 620 nm).
Table 1 and FIG. 6 (a) show the results of measuring the amount of coloration derived from anti-HP antibodies in normal (healthy) and patient sera by sandwich ELISA using CP2-PCR1 recombinant protein, respectively.
Further, Table 1 and FIG. 6 (b) show the results of measuring the amount of color derived from anti-HP antibodies in normal (healthy) and patient sera by sandwich ELISA using CP2-PCR2 recombinant protein, respectively. Show.
[0024]
[Table 1]

[0025]
As is clear from Table 1 and FIGS. 6 (a) and 6 (b), when anti-HP antibodies were measured using the recombinant CP2 antigen of the present invention, CP2-PCR1 and CP2-PCR2, all of them were patient serum and normal (healthy) 1) It is clear that there is a clear significant difference with serum.
Therefore, it can be seen that the anti-HP antibody in the serum can be detected with high sensitivity even when the sandwich ELISA method is performed using the recombinant CP2 antigen of the present invention.
[0026]
【The invention's effect】
The polypeptide of the present invention can be used as an antigen for specifically detecting an anti-HP antibody or as a vaccine against HP.
In addition, if the DNA of the present invention is used, a polypeptide that can be used as an antigen for HP-specific detection can be easily produced in large quantities by genetic engineering techniques, and an oligonucleotide can be produced using the DNA of the present invention. If a primer or a probe is prepared, HP can be detected by a PCR method or a hybridization method.
According to the method of the present invention, it is possible to easily and in large quantities obtain a polypeptide antigen that enables specific detection of HP, which has been difficult to obtain conventionally.
[0027]
[Sequence Listing]

[0028]
[SEQ ID NO: 2]

[0029]
[SEQ ID NO: 3]

[Brief description of the drawings]
FIG. 1 shows the amino acid sequence of the CP2 antigen structural gene obtained in Example 1 (SEQ ID NO: 1), the amino acid sequence of the CP2 antigen structural gene obtained in Example 4 (1) (SEQ ID NO: 2), and the results. This is a comparison of the amino acid sequence (SEQ ID NO: 3) of the CP2 antigen structural gene obtained in Example 4 (2). In the figure, * indicates that the amino acid sequence of the CP2 antigen structural gene is an amino acid in which a difference is recognized among the three.
FIG. 2 (a) shows the result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CP2-PCR1 recombinant protein obtained in Example 5. FIG.
(B) shows the results of Western blotting analysis of CP2-PCR1 recombinant protein and anti-CP2 monoclonal antibody obtained in Example 5.
FIG. 3 (a) shows the result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CP2-PCR2 recombinant protein obtained in Example 5.
(B) shows the result of Western blotting analysis of CP2-PCR2 recombinant protein and anti-CP2 monoclonal antibody obtained in Example 5.
FIG. 4 (a) shows the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CP2-PCR1 recombinant protein and CP2-PCR2 recombinant protein obtained in Example 6.
(B) shows the results of Western blotting analysis of CP2-PCR1 recombinant protein or CP2-PCR2 recombinant protein obtained in Example 6 and sera of patients with duodenal ulcer.
FIG. 5 (a) shows the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CP2-PCR1 recombinant protein and CP2-PCR2 recombinant protein obtained in Example 6.
(B) shows the results of Western blotting analysis of CP2-PCR1 recombinant protein or CP2-PCR2 recombinant protein obtained in Example 6 and normal (healthy) serum.
6 (a) shows anti-Helicobacter pylori (HP) antibody in normal (healthy person) serum and patient serum obtained by sandwich ELISA using CP2-PCR1 recombinant protein obtained in Example 7. FIG. The result of having measured the coloration amount derived from is shown.
(B) was measured by the sandwich ELISA method using CP2-PCR2 recombinant protein obtained in Example 7 and the amount of color derived from anti-HP antibody in normal (healthy) serum and patient serum. The results are shown.

Claims (7)

SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: polypeptide consisting of the amino acid sequence represented by 3. DNA encoding the polypeptide of claim 1. SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: consisting of the nucleotide sequence represented by 3 according to claim 2, wherein the DNA. A recombinant vector containing the DNA according to claim 2 or 3. A cell host transformed with the recombinant vector according to claim 4. The method for producing a polypeptide according to claim 1, wherein the cell host according to claim 5 is cultured in a medium, and the polypeptide is collected from the obtained culture. A reagent for measuring an anti-Helicobacter pylori antibody, comprising the polypeptide according to claim 1.

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