JP3814844B2 - Chlamydia pneumoniae antigen polypeptide, DNA encoding the same, recombinant vector containing the DNA, transformant containing the recombinant vector, and method for producing anti-Chlamydia pneumoniae antibody - Google Patents

Description

以下、クラミジア・ニューモニエの宿主細胞の培養から、クラミジア・ニューモニエの抗原ポリペプチドの遺伝子ＤＮＡ配列／アミノ酸配列の決定まで、順を追って説明する。
【００３５】
実施例１ クラミジア・ニューモニエ特異的５３Ｋ抗原ポリペプチドをコードするＤＮＡの作製
（Ａ）宿主細胞（ＨＬ細胞）の培養
予め、プラスチック製培養フラスコ（７５cm²）の底面いっぱいに増殖させたＨＬ細胞をリン酸緩衝化生理食塩液（以下、ＰＢＳという。）マグネシウム不含（−）液５mlで洗浄し、０．１％（ｗ／ｖ）トリプシンを含むＰＢＳを５ml加えて細胞表面全体に行き渡らせ、その液を捨てた後、３７℃で１０分間保温し、１０％（ｖ／ｖ）牛胎児血清を含むダルベッコＭＥＭ培地５mlを加え、ピペッテイングによりＨＬ細胞を剥離して、細胞浮遊液を調製した。
【００３６】
７５cm²のプラスチック製培養フラスコで培養するときは、培養フラスコに上記細胞浮遊液１ml及び１０％（ｖ／ｖ）牛胎児血清含有ダルベッコＭＥＭ培地１５〜２０mlを加え、また、６ウェルプラスチック製培養容器で培養するときは、上記細胞浮遊液８mlと１０％牛胎児血清含有ダルベッコＭＥＭ培地２９２mlとの混合液４mlずつを各ウェルに加え、５％（ｖ／ｖ）炭酸ガス雰囲気下で培養した。
【００３７】
（Ｂ）クラミジア・ニューモニエ ＹＫ４１の培養
６ウェルプラスチック製培養容器（底面上）に増殖したＨＬ細胞の培養上清をピペットで取り除き、これにクラミジア・ニューモニエＹＫ４１株（金本ら：Microbiol.Immunol.,Vol.37,P.495-498,1993）の浮遊液〔クラミジア・ニューモニエＹＫ４１保存液を、１リットルあたり庶糖７５ｇ、リン酸一カリウム０．５２ｇ、リン酸二カリウム１．２２ｇ及びグルタミン酸０．７２ｇを含む水溶液（以下、ＳＰＧという。）で１２ないし２４倍に希釈し、超音波で１分間処理し、２，０００rpmで３分間遠心分離した上清〕を１ウェルあたり２ml加えて、２，０００rpmで１時間遠心吸着を行った。遠心吸着後、クラミジア・ニューモニエ浮遊液を除き、１μg/mlシクロヘキシミド及び１０％（ｖ／ｖ）牛胎児血清を含むダルベッコＭＥＭ培地をウェルあたり４ml加え、５％（ｖ／ｖ）炭酸ガス雰囲気下、３６℃で３日間培養した。培養後、滅菌したシリコン片で細胞を剥離し、細胞を回収した。これを８，０００rpmで３０分間遠心分離して、沈殿をＳＰＧに再懸濁し、−７０℃で保存した。
【００３８】
（Ｃ）クラミジア・ニューモニエＹＫ４１の基本小体の精製
−７０℃に保存しておいたクラミジア・ニューモニエＹＫ４１感染凍結ＨＬ細胞浮遊液を融解し、テフロンホモジナイザーでホモジナイズした。２，５００rpmで１０分間遠心分離し、上清を回収した。沈殿は再びＳＰＧに懸濁し、同様の操作を行い、上清を回収した。同様の操作を更に２回行い、得られた上清は集めて合わせた。
【００３９】
別途、遠心管に５０％（ｗ／ｖ）庶糖を含む０．０３Ｍトリス−塩酸緩衝液 （pH７．４）、次いで、ウログラフィン７６％（シェーリング社製）３容量と０．０３Ｍトリス−塩酸緩衝液（pH７．４）７容量との混合液を重層し、この上に先に回収した上清を注意深く重層し、８，０００rpmで１時間遠心分離した。５０％（ｗ／ｖ）庶糖を含む０．０３Ｍトリス−塩酸緩衝液（pH７．４）層及び沈殿を回収し、この回収液に同容量のＳＰＧを加え、１０，０００rpmで３０分間遠心分離した。上清を捨て、沈殿をＳＰＧに懸濁した。遠心分離管に、ウログラフィン７６％（シェーリング社製）と０．０３Ｍトリス−塩酸緩衝液（pH７．４）の３５％から５０％（総量に対する前者の容量比）までの連続密度勾配液を作製し、この上に懸濁液を重層し、８０００rpmで１時間遠心分離した。クラミジア・ニューモニエ ＹＫ４１の基本小体に相当する黄色味を帯びた白濁したバンドを回収し、これをＳＰＧで２倍に希釈し、１００００rpmで３０分間遠心分離した。得られた沈殿をＳＰＧに懸濁し、タンパク質濃度を測定（バイオラッド社のタンパク測定キットを用い、牛血清アルブミンを標準とした）後、−７０℃で保存した。
【００４０】
（Ｄ）クラミジア・ニューモニエＹＫ−４１株のゲノムＤＮＡの調製
上記精製クラミジア・ニューモニエＹＫ−４１株の基本小体の懸濁液３００μｌ（タンパク質濃度：１．３７mg/ml）を４℃、１２，０００rpmで５分間遠心分離した。沈殿に１ｍＭ ＥＤＴＡを含む１０ｍＭトリス−塩酸緩衝液pH８．０（以下、ＴＥ緩衝液という）５００μｌを加えて懸濁した。同様の遠心分離を再度行い、沈殿を３００μｌのＴＥ緩衝液に懸濁した。１％ＳＤＳ水溶液３０μｌ及び１mg/mlプロテイナーゼＫ水溶液３０μｌを加え、５６℃で３０分間インキュベートし、基本小体を溶解させた。０．１Ｍトリス−塩酸緩衝液（pH８．０）飽和フェノール３５０μｌを加え、ボルテックスミキサーでよく混合後、４℃、１２，０００rpmで５分間遠心分離し、水層を回収した（ＤＮＡの抽出）。この抽出操作はもう一度繰り返した。１０mg/mlのＲＮａｓｅ溶液を２μｌ加え、３７℃で２時間インキュベートし、ＲＮＡを分解した。０．１Ｍトリス−塩酸緩衝液（pH８．０）飽和フェノール、クロロホルム及びイソアミルアルコールの２５：２４：１（容量比）の混合液（以下、ＰＣＩという。）３００μｌを加え、ボルテックスミキサーでよく混合し、４℃、１２，０００rpmで５分間遠心分離し、水層を回収した。この操作を合計５回繰り返した。
【００４１】
得られた液にその１／１０容の１０Ｍ酢酸アンモニウム水溶液及び２容のエタノールを加え、５分間放置し、ＤＮＡを析出させたのち、４℃、１２，０００rpmで５分間遠心分離した。沈殿は７０％エタノール水溶液６００μｌを加え、混合し、４℃、１２，０００rpmで５分間遠心分離する洗浄を２回繰り返した。遠沈管のふたを開けたまま１５分間放置して沈殿を乾燥させ、これにＴＥ２００μｌを加えて溶かし、−２０℃に保存した。
【００４２】
（Ｅ）ゲノムＤＮＡ発現ライブラリーの作製
ゲノムＤＮＡ溶液１００μｌに、制限酵素用M-buffer１０μｌ、制限酵素混合液（ＡｃｃＩ、ＨａｅIII及び１／５０希釈のＡｌｕＩ各０．４μｌとＴＥ２０μｌを混合）１０μｌを加え、３７℃で２０分間反応させた。なお、上記２０分の反応時間は、ＤＮＡが１ｋｂｐ〜７ｋｂｐの大きさの部分消化ＤＮＡに分解される時間で、予め少量のゲノムＤＮＡを用いて試験した。上記反応液にＰＣＩを１００μｌ加え、ボルテックスミキサーでよく混ぜ、４℃、１２，０００rpmで５分間遠心分離し、水層を回収した。これに３Ｍ酢酸ナトリウム水溶液１０μｌ及びエタノール２２０μｌを加え、−８０℃に１５分間静置し、部分消化ＤＮＡを析出させた。４℃、１２，０００rpmで５分間遠心分離し、上清液を捨てたのち、沈殿に７０％エタノール水溶液５００μｌを加えて混ぜ、再び、１２，０００rpmで５分間遠心分離した。上清液を捨て、沈殿を減圧下に乾燥した。
【００４３】
得られた部分消化ＤＮＡを精製水２０μｌに溶かし、その１９μｌをとり、これに下記化１で示すリンカー（２０pmole/μl）１４μｌ、１０ｍＭ ＡＴＰ４ ．５μｌ、５０ｍＭ ＭｇＣｌ₂、５０ｍＭジチオスレイトール及び５００μg/ml牛血清アルブミン含有０．２Ｍトリス−塩酸緩衝液（pH７．６、以下、１０倍濃度ライゲーション用緩衝液という）４．５μｌ、精製水２μｌ及びＴ４リガーゼ１μｌを加え、１６℃で４時間反応させ、リンカーを付加させた。
【化１】

【００４４】
リンカーを付加させた部分消化ＤＮＡを、０．１Ｍ ＮａＣｌ及び１ｍＭ ＥＤＴＡ含有１０ｍＭトリス−塩酸緩衝液を移動相とするChroma spin 6000カラムにかけた。溶出液２滴ずつを分取し、各分画の一部を０．８％アガロースゲル電気泳動で分析して、１ｋｂｐから７ｋｂｐのＤＮＡ断片を含む分画を回収した。得られた分画１４４μｌに、精製水１３μｌ、１０ｍＭ ＡＴＰ ２０μｌ、０．１Ｍ ＭｇＣｌ₂、５０ｍＭジチオスレイトール、１ｍＭスペルミジン塩酸塩及び１ｍＭ ＥＤＴＡ含有０．５Ｍトリス−塩酸緩衝液（pH７．６、以下、１０倍濃度リン酸化反応用緩衝液という。）２０μｌ、及びＴ４ポリヌクレオチドキナーゼ３μｌを加え、３７℃で３０分間反応させ、ＤＮＡ断片の５′端をリン酸化した。ＰＣＩ ２００μｌを加えてよく振り混ぜた後、４℃、１２，０００rpmで５分間遠心分離し、水層を回収した。２０mg/mlグリコーゲン水溶液１μｌ、３Ｍ酢酸ナトリウム水溶液２０μｌ及びエタノール４００μｌを加えてヌクレオチドを析出させた。４℃、１２，０００rpmで１０分間遠心分離し、上清を捨て、沈殿に７０％エタノール２００μｌを加え混ぜ、再び遠心分離し、上清を捨て、沈殿を風乾し、精製水１μｌを加え溶かした。
【００４５】
この液０．６μｌに、予め制限酵素ＥｃｏＲＩで切断したλgt11 ＤＮＡ（１μg/μl、ストラタジーン（Stratagene）社）１μｌ、１０倍濃度ライゲーション用緩衝液０．５μl、１０ｍＭ ＡＴＰ０．５μｌ、Ｔ４リガーゼ０．４μｌ及び精製水２μｌを加え、４℃で一晩反応させた。次いで、ギガパック（Gigapack）II Goldパッケージングキット（ストラタジーン社）を用い、得られた組換えλgt11ＤＮＡをパッケージングした。
【００４６】
（Ｆ）クラミジア・ニューモニエ特異的モノクローナル抗体の作製
骨髄腫細胞株の培養及び継代
モノクローナル抗体の作製に用いた骨髄腫細胞株は、Ｐ３／ＮＳＩ／１−Ａｇ４−１（ＡＴＣＣ ＴＩＢ−１８）である。１０％（ｖ／ｖ）牛胎児血清を含むＲＰＭＩ１６４０培地で培養し、継代した。細胞融合に供する２週間前に、０．１３ｍＭの８−アザグアニン、０．５μg/mlのＭＣ−２１０（マイコプラズマ除去剤、大日本製薬(株)製）及び１０％（ｖ／ｖ）牛胎児血清を含むＲＰＭＩ１６４０培地で１週間培養し、その後の１週間は通常の培地で培養した。
【００４７】
マウスの免疫
タンパク質の濃度が２７０μg/mlの上記基本小体の懸濁液２００μｌを、１２０００rpmで１０分間遠心分離し、沈殿に２００μｌのＰＢＳを加え、再懸濁した。これに２００μｌのフロイントコンプリートアジュバントを加え、エマルジョンとし、その１５０μｌをマウスの背中の皮下に注射した（この日を０日目とする）。１４日目、３４日目及び４９日目に、タンパク質の濃度が２７０μg/mlの精製基本小体の懸濁液１００μｌをマウスの腹腔内に注射した。更に、６９日目にタンパク質の濃度が８００μg/mlの精製基本小体の懸濁液５０μｌ、９２日目に同懸濁液１００μｌをマウスの腹腔内に注射し、９５日目に脾臓を取りだし、細胞融合に供した。
【００４８】
細胞融合
免疫したマウスの脾臓から得られた脾細胞１０⁸個に対して骨髄腫細胞１０⁷個を丸底ガラスチューブにとり、よく混合し、１４００rpmで５分間遠心分離し、上清を除去した後、細胞を更によく混合した。予め３７℃に保温しておいた３０％（ｗ／ｖ）ポリエチレングリコールを含むＲＰＭＩ１６４０培地０．４mlを加え、３０秒間放置した。７００rpmで６分間遠心分離した後、ＲＰＭＩ１６４０培地１０mlを加え、ポリエチレングリコールがよく混ざるようにガラスチューブをゆっくり回転させ、１４００ｒｐｍで５分間遠心分離し、上清を完全に除去し、沈殿に５mlのＨＡＴ培地を加え、５分間放置した。更に１０〜２０mlのＨＡＴ培地を加え、３０分間放置した後、骨髄腫細胞濃度が３．３×１０⁵/mlとなるようにＨＡＴ培地を加えて細胞を懸濁させ、パスツールピペットを用い９６ウェルプラスチック製培養容器のウェルに２滴ずつ分注した。５％（ｖ／ｖ）炭酸ガス雰囲気下、３６℃で培養し、１日後、７日後及び１４日後にウェルにＨＡＴ培地を１〜２滴加えた。
【００４９】
抗体生産細胞のスクリーニング
精製したクラミジアニューモニエＹＫ４１の基本小体を１％（ｗ／ｖ）ＳＤＳで可溶化し、０．０２％アジ化ソーダ含有０．０５Ｍ重炭酸ソーダ緩衝液（pH９．６）に対して透析したのち、タンパク質濃度が１〜１０μg/mlとなるように希釈した液を、塩化ビニル製９６ウェルＥＩＡ用プレートのウェルに５０μｌとり、４℃で一晩放置し、抗原を吸着させた。上澄みを除去し、ウェルに０．０２％（ｗ／ｖ）ツィーン２０を含むＰＢＳ１５０μｌを加え、３分間放置し、その後除去・洗浄した。洗浄操作を更に１回行なった後、ウェルに１％（ｖ／ｖ）牛血清アルブミンを含むＰＢＳ１００μｌを加え、４℃で一晩以上放置し、ブロッキングを行なった。牛血清アルブミンを含むＰＢＳを除いた後、０．０２％（ｗ／ｖ）ツィーン２０を含むＰＢＳで同様に２回洗浄後、ウェルに融合細胞の培養上清を５０μｌ加え、室温で２時間放置した。０．０２％（ｗ／ｖ）ツィーン２０を含むＰＢＳで同様に３回洗浄後、ウェルに２５ng/mlのペルオキシダーゼ標識化ヤギ抗マウスＩｇＧ抗体を５０μｌ加え、室温で２時間放置した。０．０２％（ｗ／ｖ）ツィーン２０を含むＰＢＳで同様に３回洗浄後、ウェルにＡＢＴＳ溶液（ＫＰＬ社製）を５０μｌ加え、室温で１５分〜１時間放置して発色反応させた後、９６ウエルＥＩＡプレート用光度計で４０５nmの吸光度を測定した。
この結果、陽性のウエルが見出され、その培養上清中には基本小体と反応する抗体が含まれていることが分かった。このウェル中の細胞をそれぞれパスツールピペットで回収し、２４ウェルプラスチック製培養容器に移し、ＨＡＴ培地１〜２ｍｌを加え、同様に培養した。
【００５０】
限界希釈法によるクローニング
２４ウェルプラスチック製培養容器で増殖させた融合細胞の細胞濃度を測定し、細胞数が２０個／mlとなるようそれぞれをＨＴ培地で希釈した。別にＨＴ培地に懸濁した４〜６週齢のマウス胸腺細胞を９６ウェルプラスチック製培養容器に２×１０⁵個／ウェルとり、これに上記の融合細胞（細胞濃度が２０個／ml）を５０μｌ／ウェルずつ加え、５％（ｖ／ｖ）炭酸ガス雰囲気下、３６℃で培養し、その１日後、７日後及び１４日後にＨＴ培地を１〜２滴／ウェル加えた。細胞の増殖が見られたウェルの培養上清を５０μｌ回収し、上記と同様の方法で抗体の生産を確認した。
ウェル中に単一の細胞コロニーしか存在せず、基本小体と反応する抗体を生産するもので、かつ増殖が早い細胞をウェルから回収し、引き続き２４ウェルプラスチック製培養容器で増殖させた。更に、同様のクローニング操作を繰り返し、最終的にハイブリドーマ、ＡＹ６Ｅ２Ｅ８を得た。
【００５１】
モノクローナル抗体の生産
ハイブリドーマＡＹ６Ｅ２Ｅ８を、１０％（ｖ／ｖ）牛胎児血清含有ＲＰＭＩ１６４０培地２０mlを入れた７５cm²プラスチック製細胞培養用フラスコで増殖させ、３〜４日ごとにその培養液から１６〜１８mlを抜き取り、代わりに新鮮な１０％（ｖ／ｖ）牛胎児血清含有ＲＰＭＩ１６４０培地を総量で２０mlとなるように補い、継代培養を続けた。抜き取って回収した細胞培養液は、１２００rpmで５分間遠心分離し、上清（モノクローナル抗体含有培養上清）を回収した。
また、予め２週間前にプリスタン０．５mlを腹腔内に注射しておいたＢａｌｂ／ｃマウスのその腹腔内に、１〜５×１０⁶個／mlとなるようＰＢＳで懸濁したハイブリドーマ株を１ml注射した。３週間後、ｂａｌｂ／ｃマウスの腹水を回収し、１２００rpmで５分間遠心分離し、上清（モノクローナル抗体含有腹水）を回収した。
【００５２】
モノクローナル抗体の精製
ハイブリドーマ ＡＹ６Ｅ２Ｅ８が生産するモノクローナル抗体は以下のようにして精製した。ハイブリドーマ ＡＹ６Ｅ２Ｅ８をマウス腹腔内に注射して得られたモノクローナル抗体含有腹水１容に３容のＰＢＳを加えて混合し、３０００rpmで１０分間遠心分離し、その上清をポアサイズ０．２２μｍのフィルタで濾過後、これをクロマトップスーパープロテインＡカラム（径４．６mm×１００mm、日本ガイシ(株)製）を用いるＨＰＬＣで精製した。カラムは予め、ＰＢＳで平衡化しておいた。
０．２２μｍフィルタで濾過後のサンプル１ｍｌをカラムに注入後、ＰＢＳを１ml/minで３分間流し、次いで、５ml/minで４分間流してカラムを洗浄した後、精製水１ＬにＮａＣｌ ８．７７ｇ、クエン酸（一水和物）１６．７ｇ及びＮａ₂ＨＰＯ₄・１２Ｈ₂Ｏ １４．７２ｇを溶かした液を２ml/minで５分間流してモノクローナル抗体を溶出した。モノクローナル抗体の溶出画分を集め、ＴＴＢＳ溶液で希釈した。
クラミジア・ニューモニエの基本小体を溶解し、基本小体に含有されているペプチドを取得した。このペプチドと上記モノクローナル抗体を用いてウェスタンブロットを行い、取得したモノクローナル抗体の特異性を確認した。
その結果、取得したモノクローナル抗体はクラミジア・ニューモニエ５３ＫＤａ抗原ポリペプチドを認識することがわかった。
ハイブリドーマ ＡＹ６Ｅ２Ｅ８と同様にして、ハイブリドーマＳＣＰ５３及びハイブリドーマ７０を取得した。上記の方法と同様にしてハイブリドーマＳＣＰ５３及びハイブリドーマ７０が産生するモノクローナル抗体の特異性を調べた結果、これらのモノクローナル抗体は、それぞれ、クラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチド及び７３ＫＤａ抗原ポリペプチドを認識することがわかった。
また、上記の方法と同様にしてハイブリドーマＳＣＰ５３及びハイブリドーマ７０が産生するモノクローナル抗体のサブクラスを調べた結果、これらの抗体のサブクラスは、それぞれ、ＩｇＧ₁及びＩｇＧであった。
【００５３】
（Ｇ）抗原ポリペプチドをコードするＤＮＡのクローニング
大腸菌Ｙ１０９０ｒ−株の一白金耳を１０ｍＭ ＭｇＳＯ₄３ml、０．２％マルトース及び５０μg/mlアンピシリン含有のＬＢ（水１Ｌ中にＮａＣｌ ５ｇ、ポリペプトン１０ｇ及び酵母エキス５ｇを含む）培地に接種し、３７℃で一晩振とう培養したのち、これを２，０００rpmで１０分間遠心分離した。沈殿（大腸菌 ）に１０ｍＭ ＭｇＳＯ₄水溶液９mlを加えて混ぜ、この大腸菌懸濁液の０．３５mlを採り、これにλgt11（ＤＮＡライブラリー）懸濁液を０．１〜１０μｌ加え、３７℃で２０分間インキューベートし、大腸菌にλgt11を感染させた。予め４７℃に保温した液状ＬＢ寒天培地２．５mlに、上記λgt11感染大腸菌を加え、これを直ちにＬＢ寒天培地上に撒いた。上層寒天培地が固化した後、４２℃で３〜４時間培養し、プラークが観察された時点で１０ｍＭ ＩＰＴＧ水溶液に浸漬したニトロセルロースフィルター（φ８２mm）を上層寒天培地に乗せ、３７℃で１２時間培養した。黒インクをつけた注射針で非対称に３ヵ所突き刺してフィルターに目印をつけた後、フィルターを寒天培地からとり出し、１５０ｍＭ ＮａＣｌ及び０．１％ツィーン２０含有２０ｍＭトリス−塩酸緩衝液（pH７．５）（以下、ＴＴＢＳ緩衝液という）で３回洗浄した。寒天培地は冷蔵庫中に保存した。
【００５４】
フィルターを１５０ｍＭ ＮａＣｌ含有２０ｍＭトリス−塩酸緩衝液(pH７．５）（以下、ＴＢＳ緩衝液という）の０．１％牛血清アルブミン含有液に浸し、３７℃で１時間振とうし、ブロッキング反応を行った。次いで、フィルターをＴＴＢＳ緩衝液で２回洗浄したのち、５〜１０μg/mlのクラミジア・ニューモニエ特異的モノクローナル抗体（ＳＣＰ５３又はAY6E2E8）のＴＴＢＳ溶液に浸し、３７℃、１時間振とうした。フィルターをＴＴＢＳ緩衝液で３回洗浄した後、パーオキシダーゼ標識の（５０ng/ml）抗マウスＩｇＧ抗体溶液（ＴＴＢＳ緩衝液）中、３７℃で１時間振とうした。フィルターをＴＴＢＳ緩衝液で３回、及びＴＢＳ緩衝液で３回洗浄した後、発色基質液（ＴＢＳ緩衝液１００mlに３０％過酸化水素水溶液６０μｌと０．３％ ４−クロロ−１−ナフトールのメタノール溶液２０mlを加えて調製）に浸漬し、室温で約３０分間放置した。十分発色した時点でフィルターをとり出し、精製水で洗浄し、風乾した。
【００５５】
フィルターの発色スポットに対応する寒天培地上のプラークを捜して同定し、この部分の寒天をパスツールピペットでつき刺し、プラークを回収した。回収したプラークはクロロホルム１滴を加えた０．１Ｍ ＮａＣｌ、８ｍＭ硫酸マグネシウム及び０．０１％ゼラチン含有５０ｍＭトリス−塩酸緩衝液（pH７．５） （以下、ＳＭ緩衝液という）中に採り、４℃で一晩放置しプラーク中のλファージを抽出した。プラークが全て上記モノクローナル抗体と反応するようになるまで、前記操作を繰り返し、抗原ポリペプチドをコードするＤＮＡをクローン化した。
このようにして、クラミジア・ニューモニエ特異的モノクローナル抗体反応性のクラミジア・ニューモニエ特異的抗原ポリペプチドを発現するλファージが得られ、これを５３−３Ｓλファージと命名した。
【００５６】
（Ｈ）５３−３Ｓλファージの培養とＤＮＡ精製
前記（Ｇ）で述べた方法と同様にしてプラークを形成させ、一つのプラークを回収し、１００μｌのＳＭ緩衝液に入れ、４℃で一晩放置しλファージを抽出した。ＬＢ培養液で一晩培養した大腸菌Ｙ１０９０ｒ−株２５０μｌに、λファージ液５〜１０μｌを加え、３７℃で２０分間放置し、大腸菌にλファージを感染させた。予め３７℃に温めておいた１０ｍＭ硫酸マグネシウムを含むＬＢ培地５０mlに接種し、λファージによる大腸菌の溶菌が起こるまで３７℃で５〜７時間振とう培養した。２５０μｌのクロロホルムを加え、３，０００rpmで１０分間遠心分離し大腸菌細胞残渣を除き、λファージ懸濁液を得た。λファージＤＮＡは、Wizard λ preps キット（プロメガ社）を用いて精製した。
【００５７】
（Ｉ）クラミジア・ニューモニエ抗原ポリペプチドをコードするＤＮＡの増幅
６００μｌ用のマイクロチューブに、精製水６１．５μｌ、１０倍濃度 反応用緩衝液（５００ｍＭ ＫＣｌ、１５ｍＭ ＭｇＣｌ₂、０．０１％ゼラチンを含むトリス−塩酸緩衝液pH８．３）１０μｌ、２０ｍＭ ｄＮＴＰ １μｌ、５３−３ＳλファージＤＮＡ溶液０．１μｌ、２０ｎＭ λgt11 forward primer（宝酒造株式会社）１μｌ、２０ｎＭ λgt11 reverse primer（宝酒造株式会社）１μｌ、AmpliTaq DNA Polymerase ０．５μｌを入れ、ミネラルオイルを２〜３滴重層した。９４℃ ３０秒、５５℃ ３０秒、７３℃ ２分のサイクルのインキュベーションを３０回繰返し、ＤＮＡを増幅した。反応後、１．２％ 低温融解アガロースゲル電気泳動を行い、増幅されたＤＮＡを切り出して Wizard PCR Prep キット（プロメガ社）で精製した。
【００５８】
（Ｊ）ＤＮＡ塩基配列分析
ＤＮＡ塩基配列分析は、ＰＣＲで増幅したＤＮＡを鋳型として、Taq DNA ポリメラーゼを用いた蛍光標識ターミネータサイクルシークエンス法でシークエンス反応を行い、３７３Ａ型ＤＮＡシークエンサ（アプライドバイオシステムズ社）で分析を行った。得られたＤＮＡ塩基配列を遺伝子配列分析ソフト「ＤＮＡＳＩＳ」（日立ソフトウェアエンジニアリング社）を用いて、編集、連結、アミノ酸翻訳領域の推定を行ない、配列番号９の配列を得た。
配列番号９の配列の解析結果から、５３ＫＤａ抗原ポリペプチドについて、そのＮ末端からＣ末端に向けて約６０％のアミノ酸配列が解明されたことが分かった。
上記クラミジア・ニューモニエ抗原ポリペプチドをコードするＤＮＡは、クラミジア・ニューモニエに特異的で、かつ、５３ＫＤａ抗原ポリペプチドを認識するモノクローナル抗体を利用してクローニングされたので、このＤＮＡは、明らかに５３ＫＤａ抗原ポリペプチドをコードしている。
配列番号９の塩基配列及びアミノ酸配列の相同性検索をＧｅｎＢａｎｋデータベースで行なった結果、高い相同性を示す既知の配列は無かった。
【００５９】
実施例２ クラミジア・ニューモニエの抗原ポリペプチドの一部を含むポリペプチドをコードするＤＮＡを含む組換えベクターの作製、及びそれを含む形質転換体の作製
前述したように、取得したＤＮＡが５３ＫＤａ抗原ポリペプチドをコードしていることが明らかであるが、念のため、下記のようにして、取得したＤＮＡを発現させ、上記抗体と反応するか否か調べた。
プラスミドｐＢＢＫ１０ＭＭを制限酵素BamHIとXhoIで切断し、１．２％低温融解アガロースゲル電気泳動を行い、約４．６ＫｂｐのＤＮＡ断片を切り出して精製した。このＤＮＡ断片100ngに、配列番号１１及び配列番号１２の合成ＤＮＡ各１ngを添加し、ＤＮＡライゲーションキット（宝酒造）を用いてこれらのＤＮＡを連結した。この反応物を大腸菌ＨＢ１０１株コンピテントセル（宝酒造）に入れ、形質転換体を作製し、プラスミドを取得し、これをｐＡＤＡ４３１と名付けた。このプラスミドを制限酵素MunIで切断した後、アルカリホスファターゼ処理し５′リン酸基を除去した。
一方、５３−３ＳλファージＤＮＡを制限酵素EcoRIで切断し、このＤＮＡ断片５０ngに、上記の制限酵素ＭｕｎＩで切断したｐＡＤＡ４３１プラスミドＤＮＡ１００ngを添加し、同様に連結し、形質転換体を作製し、５３−３ＳλファージＤＮＡの制限酵素ＥｃｏＲＩ断片が組み込まれたプラスミドを取得し、これをｐＣＰＮ５３３αと名付けた。このプラスミドは、配列番号１０の塩基配列を有する約５．７ｋｂｐのＤＮＡであり、５３Ｋ抗原ポリペプチドの一部を含むポリペプチドを宿主大腸菌で発現させることができるものである。この５３Ｋ抗原ポリペプチドの一部を含むポリペプチドをコードするＤＮＡの塩基配列は配列番号４のようになっており、この塩基配列から推定されるアミノ酸配列はを配列番号２のようになっていた。プラスミドｐＣＰＮ５３３αをもつ大腸菌を同様に培養し、電気泳動、ニトロセルロース膜への転写、モノクローナル抗体での検出を同様に行った結果、上記ポリペプチドに相当する発色したバンドが観察され、プラスミドｐＣＰＮ５３３αをもつ大腸菌が、クラミジア・ニューモニエに特異的に反応するモノクローナル抗体と反応することができる５３Ｋ抗原ポリペプチドを発現していることが示された。
【００６０】
実施例３ クラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチド全体をコードするＤＮＡの取得
配列番号９の塩基配列を元に、配列番号１４及び１５の塩基配列を有するＤＮＡを、ＤＮＡ合成機を用いて合成した。
実施例１で得たクラミジア・ニューモニエＹＫ４１株のゲノムＤＮＡの水溶液１０μｌ（ＤＮＡ含有量：約１μｇ）に、１０倍濃縮Ｋバッファ５μｌ、精製水３５μｌ及び制限酵素ＨｉｎｄIII（１９Ｕ／μｌ）５μｌを添加し、３７℃で３時間保温した。
得られた反応液をフェノールで抽出し、エタノールを添加し、遠心分離して沈殿を取得した。この沈殿に、ＰＣＲ in vitro Cloning Kit(宝酒造(株)製品名)中のＨｉｎｄIIIカセットＤＮＡ（２０ng/μl）５μｌ、ライゲーション溶液１５μｌを添加し、１６℃で３０分間保温した。
取得した反応液をフェノールで抽出し、エタノールを添加し、遠心分離して沈殿を取得し、これを１０μｌの精製水に溶解した。
得られた溶液１μｌに、精製水７８．５μｌ、１０倍濃縮ＰＣＲ用バッファ１０μｌ、２．５ｍＭｄＮＴＰ８μｌ及びＴａｑポリメラーゼ０．５μｌ（５Ｕ／μｌ）を添加し、さらに、プライマーＤＮＡとして、配列番号１４の塩基配列を有するＤＮＡ（２０pmol/μl）１μｌ及び配列番号１６の塩基配列を有するＤＮＡ（２０pmol/μl）（上記キットにおいて、プライマーＣ１として同封されていたもの）１μｌを添加して、これらを０．６ｍｌのマイクロチューブに入れ、ミネラルオイル２滴を重層し、９４℃３０秒、５５℃２分、７２℃３分の温度サイクルを３０回繰り返した。以上の工程をＰＣＲ工程という。
ＰＣＲ工程後の反応液１μｌに、プライマーＤＮＡとして、配列番号１５の塩基配列を有するＤＮＡ（２０pmol/μl）１μｌ及び配列番号１７の塩基配列を有するＤＮＡ（２０pmol/μl）（上記キットにおいて、プライマーＣ２として同封されていたもの）１μｌを用い、再度ＰＣＲ工程を行った。
２番目のＰＣＲ工程後の反応液を１．２％低融点アガロースゲル電気泳動させ、約１．４ｋｂｐの大きさのＤＮＡが含有されているアガロースゲルを切り出した。ＤＮＡの精製には Wizard PCR Prep キット（プロメガ社）を用いた。即ち 、切り出したアガロースゲルにキットに同封されている緩衝液を添加し、加熱してアガロースゲルを溶解し、キットに同封されている精製用樹脂を添加してＤＮＡを樹脂に吸着させ、遠心分離して精製用樹脂を沈殿として取得した。沈殿をプロパノールで洗浄し、再度遠心分離して沈殿を取得した。沈殿に精製水を添加し、精製用樹脂からＤＮＡを溶出して、遠心分離し、上清（ＤＮＡ水溶液）を得た。以上の工程をＤＮＡ精製工程という。
取得したＤＮＡ水溶液を用い、含まれるＤＮＡを鋳型とするTaq DNA ポリメラーゼを用いた蛍光標識ターミネータサイクルシークエンス法でシークエンス反応を行い、３７３Ａ型ＤＮＡシークエンサ（アプライドバイオシステムズ社）でそのＤＮＡの塩基配列を分析した。得られたＤＮＡ塩基配列を遺伝子配列分析ソフト「ＤＮＡＳＩＳ」（日立ソフトウェアエンジニアリング社）を用いて、編集、連結、アミノ酸翻訳領域の推定を行なった。以上の工程を塩基配列解析工程という。
取得したＤＮＡの塩基配列を解析した結果、このＤＮＡは実施例１で取得したクラミジア・ニューモニエの抗原ポリペプチドをコードするＤＮＡの中の３′末端側の約５０ｂｐの塩基配列を有していた。さらに、その塩基配列の下流には、終始コドンを含有する約０．７ｋｂのコード領域が存在していることがわかった。 配列番号９の塩基配列を元に、クラミジア・ニューモニエの抗原ポリペプチドのをコードするＤＮＡの上流部分に相当するプライマーとして、配列番号１８の塩基配列を有するＤＮＡを、また、上記の約０．７ｋｂのコード領域を含む塩基配列を元に、クラミジア・ニューモニエの抗原ポリペプチドのをコードするＤＮＡの下流部分に相当するプライマーとして、配列番号１９の塩基配列を有するＤＮＡを、それぞれ、ＤＮＡ合成機を用いて合成した。
実施例１で得たクラミジア・ニューモニエＹＫ４１株のゲノムＤＮＡの水溶液１μｌを用い、プライマーＤＮＡとして配列番号１８の塩基配列を有するＤＮＡ（２０pmol/μl）１μｌ及び配列番号１９の塩基配列を有するＤＮＡ（２０pmol/μl）１μｌを用いてＰＣＲ工程を行った。
３番目のＰＣＲ工程後の反応液を用い、上記ＤＮＡ精製工程を行い、約１．５ｋｂｐのＤＮＡを取得した。
取得したＤＮＡ水溶液を用い、上記塩基配列解析工程を行った。
取得したＤＮＡの塩基配列を解析した結果、このＤＮＡは配列番号４の塩基配列を有しており、配列番号１のアミノ酸配列をコードしていることがわかった。またプラスミドｐＣＰＮ５３３αと前述のλgt11のＤＮＡライブラリーを用いてゲノムウォーキングを行い、クラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチド全体をコードするＤＮＡを取得した。
【００６１】
実施例４ クラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチド全体をコードするＤＮＡを含む組換えベクターの作製、及びそれを含む形質転換体の作製
クラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチド全体をコードするＤＮＡを用い、実施例２と同様にしてクラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチド全体をコードするＤＮＡを含む組換えベクターとそれを含む形質転換体を作製する。
【００６２】
実施例５ クラミジア・ニューモニエの７３Ｋ抗原ポリペプチドをコードするＤＮＡの作製
モノクローナル抗体ＳＣＰ５３又はＡＹ６Ｅ２Ｅ８の代わりに、モノクローナル抗体７０を使用し、実施例１と同様の手順で操作した。クローン７０−２Ｓλファージが得られ、これから配列番号１３の配列が得られた。
配列番号１３の配列の解析結果から、クラミジア・ニューモニエの７３Ｋ抗原タンパク質については、そのＮ末端からＣ末端に向けて約９０％のアミノ酸配列が解明されたことが分かった。
配列番号１３の塩基配列及びアミノ酸配列の相同性検索をＧｅｎＢａｎｋデータベースで行なった結果、これはクラミジア・トラコマチスから単離された遺伝子塩基配列（L.M.Sardinia et al:J. Bacteriol., Vol.171, 335-341(1989)）と高い相同性を示すものであった。
【００６３】
実施例６ クラミジア・ニューモニエの抗原ポリペプチドを抗原として用いる、抗クラミジア・ニューモニエ抗体の製造
（Ａ）骨髄腫細胞株の培養及び継代
骨髄腫細胞株はＰ３Ｘ６３Ａｇ８．６５３（ＡＴＣＣ ＣＲＬ−１５８０）を１０％（ｖ／ｖ）牛胎児血清を含むＲＰＭＩ１６４０培地で培養し、継代する。細胞融合に供する２週間前に、０．１３ｍＭの８−アザグアニン、０．５μg/mlのＭＣ−２１０（マイコプラズマ除去剤、大日本製薬(株)製）及び１０％（ｖ／ｖ）牛胎児血清を含むＲＰＭＩ１６４０培地で１週間培養し、その後の１週間は通常の培地で培養する。
【００６４】
（Ｂ）マウスの免疫
タンパク質の濃度が２７０μg/mlの上記抗原ポリペプチドの懸濁液２００μｌを、１２０００rpmで１０分間遠心分離し、沈殿に２００μｌのＰＢＳを加え、再懸濁する。これに２００μｌのフロイントコンプリートアジュバントを加え、エマルジョンとし、その１５０μｌをマウスの背中の皮下に注射する（この日を０日目とする）。１４日目、３４日目及び４９日目に、タンパク質の濃度が２７０μg/mlの上記抗原ポリペプチドの懸濁液１００μｌをマウスの腹腔内に注射し、更に、６９日目にタンパク質の濃度が８００μg/mlの上記抗原ポリペプチドの懸濁液５０μｌ、９２日目に同懸濁液１００μｌをマウスの腹腔内に注射し、９５日目に脾臓を取り出し、細胞融合に供する。
【００６５】
（Ｃ）細胞融合
上記脾臓から得られる脾細胞１０⁸個に対して骨髄腫細胞１０⁷個を丸底ガラスチューブにとり、よく混合し、１４００rpmで５分間遠心分離し、上清を除去し 、細胞を更によく混合する。予め３７℃に保温してある３０％（ｗ／ｖ）ポリエチレングリコールを含むＲＰＭＩ１６４０培地０．４mlを加え、３０秒間放置する。７００rpmで６分間遠心分離した後、ＲＰＭＩ１６４０培地１０mlを加え、ポリエチレングリコールがよく混ざるようにガラスチューブをゆっくり回転させ、１４００rpmで５分間遠心分離し、上清を完全に除去し、沈殿に５mlのＨＡＴ培地を加え、５分間放置する。更に１０〜２０mlのＨＡＴ培地を加え、３０分間放置し、骨髄腫細胞濃度が３．３×１０⁵/mlとなるようにＨＡＴ培地を加えて細胞を懸濁させ、パスツールピペットを用い９６ウェルプラスチック製培養容器のウェルに２滴ずつ分注する。５％（ｖ／ｖ）炭酸ガス雰囲気下、３６℃で培養し、１日後、７日後及び１４日後にウェルにＨＡＴ培地を１〜２滴加える。
【００６６】
（Ｄ）抗体生産細胞のスクリーニング
上記抗原ポリペプチドをタンパク質濃度が１〜１０μg/mlとなるように０．０２％（ｗ／ｖ）アジ化ソーダ含有０．０５Ｍ重炭酸ソーダ緩衝液（pH９．６）に懸濁し、０．０２％アジ化ソーダ含有０．０５Ｍ重炭酸ソーダ緩衝液（pH９．６）に対して透析し、その後、タンパク質濃度が１〜１０μg/mlとなるように希釈した液を、塩化ビニル製９６ウェルＥＩＡ用プレートのウェルに５０μｌとり、４℃で一晩放置し、抗原を吸着させる。上澄みを除去し、ウェルに０．０２％ （ｗ／ｖ）ツィーン２０を含むＰＢＳ１５０μｌを加え、３分間放置し、その後除去・洗浄する。洗浄操作を更に１回行なった後、ウェルに１％（ｖ／ｖ）牛血清アルブミンを含むＰＢＳ１００μｌを加え、４℃で一晩以上放置し、ブロッキングを行なう。牛血清アルブミンを含むＰＢＳを除いた後、０．０２％（ｗ／ｖ）ツィーン２０を含むＰＢＳで同様に２回洗浄後、ウェルに融合細胞の培養上清を５０μｌ加え、室温で２時間放置する。０．０２％（ｗ／ｖ）ツィーン２０を含むＰＢＳで同様に３回洗浄後、ウェルに２５ng/mlのペルオキシダーゼ標識化ヤギ抗マウスＩｇＧ抗体を５０μｌ加え、室温で２時間放置する。０．０２％ （ｗ／ｖ）ツィーン２０を含むＰＢＳで同様に３回洗浄後、ウェルにＡＢＴＳ溶液（ＫＰＬ社製）を５０μｌ加え、室温で１５分〜１時間放置して発色反応させ、９６ウエルＥＩＡプレート用光度計で４０５nmの吸光度を測定する。そして陽性のウエル中の細胞をそれぞれパスツールピペットで回収し、２４ウェルプラスチック製培養容器に移し、ＨＡＴ培地１〜２mlを加え、同様に培養する。
【００６７】
（Ｅ）限界希釈法によるクローニング
２４ウェルプラスチック製培養容器で増殖させた２株の融合細胞の細胞濃度を測定し、細胞数が２０個/mlとなるようそれぞれをＨＴ培地で希釈する。別にＨＴ培地に懸濁した４〜６週齢のマウス胸腺細胞を９６ウェルプラスチック製培養容器に１〜２×１０⁵個/ウェルとり、これに上記の融合細胞（細胞濃度が２０個/ml）を５０μl/ウェルずつ加え、５％（ｖ／ｖ）炭酸ガス雰囲気下、３６℃で培養し、その１日後、７日後及び１４日後にＨＴ培地を１〜２滴／ウェル加える。細胞の増殖が見られたウェルの培養上清を５０μｌ回収し、上記（Ｄ）の「抗体生産細胞のスクリーニング」と同様の方法で抗体の生産を確認する。
ウェル中に単一の細胞コロニーしか存在せず、基本小体と反応する抗体を生産するもので、かつ増殖が早い細胞をウェルから回収し、引き続き２４ウェルプラスチック製培養容器で増殖させる。更に、同様のクローニング操作を繰り返し、抗クラミジア・ニューモニエ抗体を産生するハイブリドーマを取得する。これを培養し、その培養上清から抗クラミジア・ニューモニエ抗体を製造する。
【００６８】
【発明の効果】
請求項１記載の抗原ポリペプチドは、クラミジア・ニューモニエの抗体検査等に利用できる。
請求項２記載の抗原ポリペプチドは、請求項１記載の抗原ポリペプチドの効果を奏し、さらに、アミノ酸配列の長さが短いため、担体等に固定化できる抗原ペプチドの数を多くすることができ、それにより、感度の高い診断薬の製造に利用できる。
請求項３記載の抗原ポリペプチドは、請求項１記載の抗原ポリペプチドの効果を奏し、さらに、タンパク質分解酵素による分解を受けにくい構造をつくることができるので、抗原として安定性に優れる。
請求項４記載の抗原ポリペプチドは、請求項１記載の抗原ポリペプチドの効果を奏し、さらに、アミノ酸若しくは２〜１０００個のアミノ酸配列を利用して担体等に固定化できるので、固定化による抗原性の低下又は喪失が生じにくい。
請求項５記載の抗原ポリペプチドは、請求項１記載の抗原ポリペプチドの効果を奏し、さらに、クラミジア・ニューモニエに特異的な抗原ポリペプチドの全体を有するので、抗体検査やクラミジア・ニューモニエ感染の正確な診断に極めて適切である。
請求項６記載の抗原ポリペプチドは、請求項１記載の抗原ポリペプチドの効果を奏し、さらに、クラミジア・ニューモニエに特異的な抗原部分を有するので、抗体検査やクラミジア・ニューモニエ感染の正確な診断に極めて適切である。
請求項７記載の抗原ポリペプチドは、請求項１記載の抗原ポリペプチドの効果を奏し、さらに、クラミジア・ニューモニエに特異的な抗原部分を有するので、抗体検査やクラミジア・ニューモニエ感染の正確な診断に極めて適切である。
請求項８記載のＤＮＡは、クラミジア・ニューモニエの抗体検査やクラミジア・ニューモニエ感染の診断等に好適な抗原ポリペプチドの製造に利用できる。
請求項９記載のＤＮＡは、請求項８記載のＤＮＡの効果を奏し、さらに、このＤＮＡにコードされている抗原ポリペプチドはクラミジア・ニューモニエに特異的な抗原ポリペプチドの全体を有するので、クラミジア・ニューモニエの特異的抗体検査等に好適な抗原ポリペプチドの製造に利用できる。
請求項１０記載のＤＮＡは、請求項８記載のＤＮＡの効果を奏し、さらに、このＤＮＡにコードされている抗原ポリペプチドはクラミジア・ニューモニエに特異的な抗原部分を有するので、クラミジア・ニューモニエの特異的抗体検査等に好適な抗原ポリペプチドの製造に利用できる。
請求項１１記載のＤＮＡは、請求項８記載のＤＮＡの効果を奏し、さらに、このＤＮＡにコードされている抗原ポリペプチドはクラミジア・ニューモニエに特異的な抗原部分を有するので、クラミジア・ニューモニエの特異的抗体検査等に好適な抗原ポリペプチドの製造に利用できる。
請求項１２記載の組換えベクターは、クラミジア・ニューモニエの抗体検査やクラミジア・ニューモニエの感染症の診断に好適な抗原ポリペプチドの製造に利用できる。
請求項１３記載の組換えベクターは、請求項１２記載の組換えベクターの効果を奏し、さらに、クラミジア・ニューモニエに特異的な抗原部分を有するポリペプチドを発現させることができるので、クラミジア・ニューモニエの特異的抗体検査等に極めて適切な抗原ポリペプチドの製造に利用できる。
請求項１４記載の形質転換体は、クラミジア・ニューモニエの特異的抗体検査等に好適な抗原ポリペプチドの製造に利用できる。
請求項１５記載の抗クラミジア・ニューモニエ抗体の製造方法は、クラミジア・ニューモニエ感染の診断薬製造に利用できる。
【００６９】
【配列表】

【００７０】

【００７１】

【００７２】

【００７３】

【００７４】

【００７５】

【００７６】

【００７７】

【００７８】

【００７９】

【００８０】

【００８１】

【００８２】

【００８３】

【００８４】

【００８５】

【００８６】

【００８７】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an antigen polypeptide of Chlamydia pneumoniae useful for diagnosis of Chlamydia pneumoniae infection, a DNA encoding the same, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and an anti-Chlamydia pneumoniae The present invention relates to a method for producing a Pneumonier antibody. The present invention is effectively used in the pharmaceutical industry, particularly in the manufacture of diagnostic agents for Chlamydia pneumoniae infections.
[0002]
[Prior art]
Bacteria belonging to the genus Chlamydia are known to have species (Species) such as Chlamydia trachomatis, Chlamydia sshitasi, Chlamydia pneumoniae. Chlamydia trachomatis is a causative agent that causes trachoma, sexually transmitted lymphogranulomas, genitourinary infections, inclusion body conjunctivitis, neonatal pneumonia, etc. Chlamydia sshitasi is a causative agent of parrot disease, etc. Is a causative bacterium such as respiratory infection, atypical pneumonia.
[0003]
Symptoms of respiratory infections caused by Chlamydia pneumoniae are often misdiagnosed because they are similar to those of infections caused by Mycoplasma pneumoniae and influenza viruses. Therefore, development of a simple diagnostic method for Chlamydia pneumoniae has been desired.
[0004]
The diagnosis of infectious diseases is usually made deterministic by detecting the presence of causative bacteria at the site of infection or the like, or detecting the presence of antibodies (against the causative bacteria) in serum and other body fluids. The former is called an antigen test and the latter is called an antibody test, both of which have important clinical significance.An antibody test for Chlamydia pneumoniae is a method that detects the presence of antibodies using the basic body of Chlamydia pneumoniae. Are known.
[0005]
[Problems to be solved by the invention]
However, the basic body of Chlamydia pneumoniae contains antigens that are also present in Chlamydia pneumoniae other than Chlamydia pneumoniae, that is, Chlamydia trachomatis or Chlamydia sshitasi. It reacts not only with antibodies against pneumoniae but also with antibodies against other types of Chlamydia, and there is a difficulty in lacking specificity. The present invention provides an antigen polypeptide that reacts only with a Chlamydia pneumoniae-specific antibody and does not react with antibodies against Chlamydia pneumoniae bacteria other than Chlamydia pneumoniae such as Chlamydia trachomatis and Chlamydia scitasci The purpose is to do. Furthermore, an object of the present invention is to provide an antigen polypeptide for detecting an antibody that reacts in common with Chlamydia bacteria such as Chlamydia pneumoniae, Chlamydia trachomatis, and Chlamydia schitasci.
[0006]
[Means for Solving the Problems]
The inventors of the present invention purely used a Chlamydia pneumoniae-specific antigen polypeptide or a Chlamydia spp. Specific (ie, an antigen polypeptide common to Chlamydia spp. In order to obtain the amino acid sequence in an elucidated form, first cultivate Chlamydia pneumoniae in the host cell, extract genomic DNA from the Chlamydia pneumoniae, partially digest it with restriction enzymes, and insert it into λgt11 DNA. A genomic DNA library is prepared, and this is infected with E. coli Y1090r-strain, and the colony of infected E. coli expressing the antigenic polypeptide of Chlamydia pneumoniae using a Chlamydia pneumoniae-specific monoclonal antibody or Chlamydia spp. Λ phage was extracted from positively infected E. coli, and this procedure was repeated to purify λ phage, which was then amplified by infecting E. coli Y1090r- strain, and then analyzing the base sequence of the DNA, This was translated into a polypeptide, the amino acid sequence of the antigen polypeptide was determined, and the present invention was completed.
[0007]
The present invention relates to the following (1) to (15).
(1) An antigen polypeptide of Chlamydia pneumoniae comprising polypeptide A comprising at least 5 consecutive amino acid sequences in the polypeptide of SEQ ID NO: 1.
(2) The antigen polypeptide according to (1) above, wherein the polypeptide A is a polypeptide lacking an amino acid from the polypeptide of SEQ ID NO: 1.
(3) Polypeptide A is a polypeptide in which an amino acid in the polypeptide of SEQ ID NO: 1 is substituted with another amino acid or an amino acid is inserted into the polypeptide of SEQ ID NO: 1 1) The antigen polypeptide as described.
(4) The antigen polypeptide according to (1) above, wherein the polypeptide A is a polypeptide in which an amino acid or peptide is bound to at least 5 consecutive amino acid sequences in the polypeptide of SEQ ID NO: 1.
(5) The antigen polypeptide according to (1) above, wherein the polypeptide A is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.
(6) The antigen polypeptide according to (1) above, wherein the polypeptide A is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.
(7) The antigen polypeptide according to (1) above, wherein the polypeptide A is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5.
(8) DNA encoding the antigen polypeptide according to any one of (1) to (7) above or DNA complementary thereto.
(9) The DNA according to (8) above, wherein the base sequence is the base sequence of SEQ ID NO: 3.
(10) The DNA according to (8) above, wherein the base sequence is the base sequence of SEQ ID NO: 4.
(11) The DNA according to (8) above, wherein the base sequence is the base sequence of SEQ ID NO: 7.
(12) A recombinant vector comprising the DNA according to any one of (8) to (11) above.
(13) The recombinant vector according to (12) above, wherein the recombinant vector is a pCPN533α plasmid having the base sequence of SEQ ID NO: 10.
(14) A transformant comprising the recombinant vector according to (12) or (13).
(15) A method for producing an anti-Chlamydia pneumoniae antibody, wherein the antigen polypeptide according to any one of (1) to (7) above is used as an antigen.
[0008]
The present invention also relates to the following (16) to (22).
(16) (a) the polypeptide of SEQ ID NO: 5;
(b) a polypeptide lacking one or more of the amino acids in the polypeptide of SEQ ID NO: 5;
(c) a polypeptide in which one or more of the amino acids in the polypeptide of SEQ ID NO: 5 are substituted with other amino acids; and
(d) a fusion polypeptide obtained by binding another amino acid or peptide to any one of the polypeptides (a) to (c) above,
An antigenic polypeptide of Chlamydia pneumoniae selected from the group consisting of
[0009]
(17) (a) the polypeptide of SEQ ID NO: 6;
(b) a polypeptide lacking one or more of the amino acids in the polypeptide of SEQ ID NO: 6;
(c) a polypeptide in which one or more of the amino acids in the polypeptide of SEQ ID NO: 6 are substituted with other amino acids; and
(d) a fusion polypeptide obtained by binding another amino acid or peptide to any one of the polypeptides (a) to (c) above,
An antigenic polypeptide of Chlamydia pneumoniae selected from the group consisting of
[0010]
(18) DNA encoding the polypeptide of (16) above or DNA complementary thereto.
(19) DNA encoding the polypeptide of (17) above or DNA complementary thereto.
(20) The DNA of (18) above, wherein the DNA encoding the polypeptide of (16) is SEQ ID NO: 7.
(21) The DNA of (19) above, wherein the DNA encoding the polypeptide of (17) is SEQ ID NO: 8.
(22) A recombinant vector comprising the DNA of any one of (18) to (21) above.
[0011]
In the present specification, deoxynucleotides having 1 base are referred to as monodeoxynucleotides, and deoxynucleotides having 2 or more bases are collectively referred to as DNA unless otherwise specified.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
1. Antigenic polypeptide
The antigen polypeptide of the present invention is a polypeptide comprising at least 5 consecutive amino acid sequences in the polypeptide of SEQ ID NO: 1 (hereinafter, “polypeptide A”) from the viewpoint of the minimum size of the peptide having antigenicity. It is made up of).
Since a highly sensitive antigen-antibody reaction can be expected with a longer amino acid sequence, the polypeptide A is preferably 20 or more, more preferably 100 or more, and even more preferably 250 or more amino acids.
Moreover, as long as it has antigenicity as Chlamydia pneumoniae, polypeptide A may be a polypeptide lacking amino acids (for example, 1 to 250) from the polypeptide of SEQ ID NO: 1. If the number of amino acids to be deleted is too large, the antigenicity of polypeptide A as Chlamydia pneumoniae tends to be impaired.
[0013]
When the number of amino acids to be deleted is large (for example, 5 or more), in order to maintain the antigenicity as Chlamydia pneumoniae, polypeptide A is one in which amino acids are continuously deleted (for example, 5 or more). Preferably there is.
Moreover, as long as it has antigenicity as Chlamydia pneumoniae, the polypeptide A is one in which the amino acid (for example, 1 to 100) in the polypeptide of SEQ ID NO: 1 is substituted with another amino acid. Alternatively, an amino acid (for example, 1 to 100) may be inserted into the polypeptide of SEQ ID NO: 1. When too many amino acids are substituted or inserted, the antigenicity of polypeptide A as Chlamydia pneumoniae tends to be impaired. When the number of amino acids to be substituted or inserted is large (for example, 5 or more), in order to maintain the antigenicity as Chlamydia pneumoniae, polypeptide A is continuously substituted or inserted for amino acids (for example, 5 or more). It is preferable that The amino acid to be substituted preferably has similar properties, for example, glycine and alanine substitution.
[0014]
In addition, as long as it has antigenicity as Chlamydia pneumoniae, as polypeptide A, at least five consecutive amino acid sequences in the polypeptide of SEQ ID NO: 1, directly or via an intervening amino acid sequence, A polypeptide to which an amino acid or peptide is bound may be used.
In order to maintain the antigenicity of Chlamydia pneumoniae, such a peptide is preferably composed of 1000 or less amino acid sequences, more preferably composed of 500 or less amino acid sequences, and 200 or less amino acid sequences. More preferably, it consists of
Examples of such amino acids or peptides include leucine, leucine-methionine, dihydrofolate reductase (DHFR), β-galactosidase and the like.
The intervening amino acid sequence is not particularly limited, and examples thereof include leucine and leucine-methionine amino acid sequences.
Specific examples of the polypeptide A include, for example, the polypeptides of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5.
[0015]
An example of a polypeptide lacking 1 to 250 amino acids from the polypeptide of SEQ ID NO: 1 is the polypeptide of SEQ ID NO: 5.
The polypeptide of SEQ ID NO: 1 of the present invention is an antigen polypeptide consisting of 488 amino acid residues as shown in the sequence listing.
The polypeptide of SEQ ID NO: 2 of the present invention is an antigen polypeptide consisting of 271 amino acid residues as shown in the sequence listing.
The polypeptide of SEQ ID NO: 5 of the present invention is an antigen polypeptide consisting of 259 amino acid residues as shown in the sequence listing.
The polypeptide of SEQ ID NO: 6 according to the present invention is an antigen polypeptide consisting of 571 amino acid residues as shown in the sequence listing.
Among the above antigen polypeptides, the polypeptide of SEQ ID NO: 1 containing the entire Chlamydia pneumoniae 53 KDa antigen polypeptide is desirable.
[0016]
2. Method for producing antigen polypeptide
Methods for producing the antigen polypeptide of the present invention include chemical synthesis methods and gene recombination methods.
As a chemical synthesis method, for example, there is a map (Multiple Antigen Peptide, MAP) method, which is suitable for the synthesis of peptides consisting of 30 or less amino acid sequences, and can be synthesized using a commercially available peptide synthesizer. .
As a genetic recombination method, for example, a DNA encoding the antigen polypeptide of the present invention is inserted into a vector to construct a recombinant vector, which is inserted into a host to produce a transformant, and the transformant There is a method for purifying a target peptide from
The DNA encoding the antigen polypeptide of the present invention will be described later.
Examples of vectors include plasmids and phages.
Examples of the host include Escherichia coli, Bacillus subtilis, and yeast.
Hereinafter, a method for producing a transformant and a method for purifying a target peptide using the transformant will be described in detail.
[0017]
Production of recombinant vector containing DNA encoding antigen polypeptide and production of transformant containing the same
The λ phage itself obtained by screening (described later) is also a recombinant vector containing the DNA of the present invention. However, the DNA encoding the Chlamydia pneumoniae antigen polypeptide (described later) can be converted into an existing plasmid vector or phage vector by a conventional method. A new recombinant vector can also be produced by insertion. At that time, a linker is used if necessary. As an existing plasmid vector, for example, pBR322, pUC18, pUC19, pBBK10MM and the like can be used. pBR322, pUC18, and pUC19 are commercially available, and pBBK10MM is described in detail in Japanese Patent Application Laid-Open No. 4-117284. Deposited at the institute. As phage vectors, λgt11 phage, λgt10 phage, and the like can be used. In either case, a recombinant vector corresponding to the parent vector used is obtained.
Examples of the recombinant vector containing the DNA of the present invention include pCPN533α plasmid, 53-3Sλ phage and the like as described later.
The obtained recombinant vector is put into a host to produce a transformant. When using E. coli-derived plasmids or λ phages, E. coli can be used as the host, for example, E. coli HB101 strain can be used. This host is treated to become competent cells. Competent cells obtained by treating E. coli strain HB101 are sold by Takara Shuzo. A method for preparing a transformant by putting the ligation reaction product into a host is described in the document “Molecular Cloning” (described later).
[0018]
The resulting transformant is cultured to form colonies, plasmid DNA is obtained from each colony, cut with an appropriate restriction enzyme, analyzed by agarose gel electrophoresis, and a transformant having the desired recombinant plasmid Select. An example of the plasmid vector thus prepared is pCPN533α plasmid. An example of the transformant thus prepared is Escherichia coli HB101 containing the aforementioned recombinant vector pCPN533α.
In the culture of the transformant, the incubator is shaken at an appropriate temperature until the antigen polypeptide is sufficiently accumulated in the transformant in a medium in which the transformant can grow. When using Escherichia coli HB101 strain containing the above-described recombinant vector pCPN533α as a transformant, the culture is shaken overnight at 37 ° C. in LB medium containing ampicillin, and then this culture solution is TB medium containing ampicillin or the like. And incubate overnight at 37 ° C with shaking. The preparation method of TB medium is described in the document “Molecular Cloning” (described later).
[0019]
When disrupting the cultured transformant, the transformant is collected by centrifugation, suspended in a buffer solution, and irradiated with ultrasonic waves. When the transformant is Escherichia coli, lysozyme may be added to the above suspension, and the cells may be lysed by adding a buffer containing SDS.
On the other hand, when the target polypeptide is secretable, the culture solution is centrifuged to obtain a supernatant.
The transformant is disrupted or dissolved, and then centrifuged to remove cell debris and obtain a supernatant.
Streptomycin sulfate is added to any of the above supernatants, stirred for a while, and centrifuged to remove nucleic acids as a precipitate and obtain a supernatant.
The supernatant is precipitated with ammonium sulfate and centrifuged. Usually, the precipitate is obtained, but the target peptide may be contained in the supernatant, and the presence of the target peptide is confirmed by sampling.
The above precipitate is dissolved in a small amount of buffer or the supernatant is fractionated by liquid chromatography, and the protein contained in each fraction is subjected to Western blotting using the aforementioned Chlamydia pneumoniae-specific monoclonal antibody. To obtain a fraction containing the antigen polypeptide. Specific methods of removal of residues such as cell membranes, removal of DNA to which streptomycin sulfate is added, acquisition of proteins to which ammonium sulfate is added, and Western blotting are described in the document "Molecular Cloning" (described later). .
[0020]
3. DNA encoding an antigen polypeptide
In the present invention, DNA encoding the polypeptide of SEQ ID NO: 1 refers to the amino acid according to the triplet code table (1 to 6 nucleotide sequences are assigned to each amino acid) of the polypeptide of SEQ ID NO: 1. Is a DNA selected from the group of DNAs (including the DNA of SEQ ID NO: 3).
The DNA encoding the antigen polypeptide is a DNA encoding the polypeptide A, and this DNA is selected from the DNA group obtained by replacing the amino acid sequence of the polypeptide A with the nucleotide sequence according to the triplet code table. That is.
Examples of the polypeptide A include those described in the above-mentioned antigen polypeptide section. The DNA encoding the polypeptide A also has a nucleotide sequence corresponding to the amino acid sequence of those polypeptides.
Similarly, in the present invention, the DNA encoding the polypeptide of SEQ ID NO: 2 refers to the triplet code table for the polypeptide of SEQ ID NO: 2 (1 to 6 nucleotide sequences are assigned to each amino acid). ) Is a DNA selected from a group of DNAs when amino acids are read as nucleotide sequences according to (1) (including the DNA of SEQ ID NO: 4).
The DNA encoding the polypeptide of SEQ ID NO: 5 is a group of DNAs obtained by replacing the polypeptide of SEQ ID NO: 5 with a nucleotide sequence according to the triplet code table (this includes the DNA of SEQ ID NO: 7). DNA selected from
The DNA encoding the polypeptide of SEQ ID NO: 6 is a DNA group obtained by replacing the amino acid of the polypeptide of SEQ ID NO: 6 with the nucleotide sequence according to the triplet code table (this includes the DNA of SEQ ID NO: 8). DNA selected from
[0021]
DNA encoding polypeptide A can be prepared by chemical synthesis or genetic recombination.
As a chemical synthesis method, for example, there is a phosphoamidide method, which is suitable for the synthesis of DNA consisting of a base sequence having a total length of 100 bases or less, and can be chemically synthesized with a commercially available DNA synthesizer.
Examples of the gene recombination method include a method of cloning DNA from the basic body of Chlamydia pneumoniae as described later, or a method based on the base sequence at an arbitrary position of the DNA using already obtained DNA as a template. PCR method using the prepared primers. The gene recombination method can also produce a long DNA of 100 bases or more.
Next, a method for cloning DNA encoding an antigen polypeptide from the basic body of Chlamydia pneumoniae will be described in detail.
[0022]
Chlamydia pneumoniae culture
A cell suspension is prepared from cultured HL cells and the like, and after removing the culture supernatant, a suspension of Chlamydia pneumoniae is added and cultured, and centrifuged to obtain Chlamydia pneumoniae-infected HL cells. Examples of Chlamydia pneumoniae include Chlamydia pneumoniae strain YK41 (Kanemoto et al .: Microbiological Immunology, 37, 495-498, 1993 (Y. Kanamoto et al., Microbiol. Immunol., Vol. 37, p.495-498, 1993)) can be used.
[0023]
Purification of the basic body of Chlamydia pneumoniae
Chlamydia pneumoniae-infected HL cells are disrupted, centrifuged and the supernatant is recovered. The supernatant is added to a continuous density gradient solution using urografin (manufactured by Schering) and centrifuged. The preliminary experiment confirmed that the basic body of Chlamydia pneumoniae was contained in the yellowish white band, and this band was collected.
[0024]
Preparation of Chlamydia pneumoniae genomic DNA
The basic body of Chlamydia pneumoniae is suspended in 10 mM Tris-HCl buffer (pH 8.0) (hereinafter referred to as TE buffer) containing 1 mM ethylenediaminetetraacetic acid (EDTA), and 1% sodium dodecyl sulfate (SDS). An aqueous solution and a 1 mg / ml proteinase K aqueous solution are added and kept warm to dissolve the basic bodies. Add 0.1M Tris-HCl buffer (pH 8.0) saturated phenol, stir, centrifuge, and recover the aqueous layer. Further, RNAse (RNase) treatment is performed, phenol / chloroform / isoamyl alcohol treatment and ethanol precipitation treatment are performed to obtain Chlamydia pneumoniae genomic DNA.
[0025]
Preparation of genomic DNA expression library
Genomic DNA is digested with restriction enzymes AccI, HaeIII and AluI, and subjected to phenol / chloroform / isoamyl alcohol treatment and ethanol precipitation treatment to obtain partially digested DNA. A linker, adenosine-5'-triphosphate (hereinafter abbreviated as ATP) and T4 ligase are added to the partially digested DNA to add a linker to the partially digested DNA.
This is applied to a Chroma spin 6000 column using 10 mM Tris-HCl buffer containing 0.1 M NaCl and 1 mM EDTA as a mobile phase, and the eluate is collected to obtain a DNA fragment containing 1 kbp to 7 kbp. Collect the picture. To the obtained fraction, ATP and T4 polynucleotide kinase are added and reacted to phosphorylate the 5 ′ end of the DNA fragment. The reaction solution is treated with phenol / chloroform / isoamyl alcohol and precipitated with ethanol to obtain a DNA fragment phosphorylated at the 5 'end.
This DNA fragment was reacted with λgt11 DNA, ATP and T4 ligase, which had been cleaved with restriction enzyme EcoRI in advance, and the resulting recombinant λgt11 DNA was packaged using a commercially available packaging kit. Make a rally.
[0026]
Cloning of DNA encoding antigenic polypeptide
E. coli Y1090r- strain culture medium is infected with the genomic DNA expression library, cultured on an agar medium, and immersed in an aqueous isopropylthio-β-D-galactoside (IPTG) solution using a nitrocellulose filter. The protein produced in the bacterial body by the expression of is attached to the nitrocellulose filter. The filter is blocked with bovine serum albumin, washed, and then the filter is reacted with a Chlamydia pneumoniae specific monoclonal antibody. As a Chlamydia pneumoniae-specific monoclonal antibody, for example, AY6E2E8 or SCP53 can be used. The hybridoma producing AY6E2E8 has been deposited at the Biotechnology Institute of Industrial Technology, Accession No. FERM BP-5154. For the hybridoma producing SCP53, see Journal of Clinical Microbiology, 132, pp. 583-588 (1994) (J. Clin. Microbiol., Vol. 132, p.583-588, 1994). Are listed. After the reaction, the filter is washed and reacted with an anti-mouse IgG antibody labeled with an enzyme such as peroxidase. After the reaction, the filter is washed, and a coloring substrate solution is added to react. As the coloring substrate solution, for example, a solution containing an aqueous hydrogen peroxide solution and a methanol solution of 4-chloro-1-naphthol can be used. After the reaction, the filter is washed and air dried.
[0027]
Plaques on the agar medium corresponding to the colored spots of the filter are identified, and λ phage contained in the plaques is obtained. The above procedure is repeated until all the plaques react with the monoclonal antibody, and the DNA encoding the antigen polypeptide is cloned to express the Chlamydia pneumoniae-specific monoclonal antibody-reactive Chlamydia pneumoniae-specific antigen polypeptide. Obtain λ phage.
[0028]
Obtaining DNA encoding Chlamydia pneumoniae antigen polypeptide
The obtained λ phage is infected with E. coli Y1090r− strain and cultured to produce a large amount of λ phage. DNA is obtained and purified from λ phage using a commercially available kit. Primers, tack polymerase (Taq Polymerase), and deoxynucleotides are added to this DNA, and the steps of heating, cooling, and heat insulation are repeated to amplify the DNA inserted into λgt11. Examples of the primer include λgt11 forward primer and λgt11 reverse primer (both manufactured by Takara Shuzo Co., Ltd.). There is a polymerase (AmpliTaq DNA Polymerase). The general method of this DNA amplification method is known as a PCR method. For details, see “Sam Block et al., Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory) (1989)” (J. Samblook et al., Molecular Cloning 2nd ed., Cold Spring Harbor Laboratory Press (1989), hereinafter this document is described in the document "Molecular Cloning".
[0029]
Obtain the amplified DNA and determine and analyze the base sequence. A commercially available kit can be used for obtaining DNA, for example, Wizard PCR Prep kit (product of Promega) can be used. In addition, the nucleotide sequence can be determined by a fluorescence-labeled terminator cycle sequence method using tack polymerase. To use this method, a kit sold by Perkin Elmer Japan can be used. For analysis, a commercially available machine such as a 373A DNA sequencer (Applied Biosystems) can be used.
[0030]
After the base sequence is determined, the obtained DNA base sequence is analyzed with gene sequence analysis software, and editing, linking, and amino acid translation region estimation are performed. As the gene sequence analysis software, “DNASIS” (Hitachi Software Engineering Co., Ltd.) can be used.
As a result of analysis, when a full-length gene cannot be obtained, DNA before and after the already obtained DNA is obtained by genome walking. To perform genome walking, a kit sold by Takara Shuzo Co., Ltd. can be used.
[0031]
4). Method for producing anti-Chlamydia pneumoniae antibody using antigen polypeptide as antigen
In order to produce an anti-Chlamydia pneumoniae antibody, a mouse is immunized with the antigen polypeptide of the present invention as an antigen, and its spleen cells are fused with a myeloma cell line to produce a hybridoma from which 53 KDa of Chlamydia pneumoniae is produced. It can be obtained by selecting a hybridoma that recognizes the antigen polypeptide and culturing it.
As the myeloma cell line, for example, P3X63Ag8.653 (ATCC CRL-1580) or P3 / NSI / 1-Ag4-1 (ATCC TIB-18) can be used.
An anti-Chlamydia pneumoniae antibody is produced according to a known general method for obtaining an antibody by immunizing a mouse except that the antigen polypeptide of the present invention is used as an antigen.
[0032]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited at all by this.
[0033]
In the following examples, the monoclonal antibodies used are SCP53, AY6E2E8 and 70. SCP53 and 70 are secreted by hybridoma SCP53 and hybridoma 70 obtained by immunization of a mouse by one of the present inventors Matsumoto et al. Using Chlamydia pneumoniae KKpn-1 strain as an antigen and fusing the spleen cells with myeloma cells. In addition, AY6E2E8 was obtained by one of the inventors of the present inventor who immunized mice using the basic body of Chlamydia pneumoniae YK-41 as an antigen and fused the spleen cells with myeloma. Antibody AY6E2E8 secreted by the resulting hybridoma AY6E2E8.
As shown in Table 1, monoclonal antibodies SCP53 and AY6E2E8 are specific for C. pneumoniae, and monoclonal antibody 70 is specific for Chlamydia bacteria. A method for producing a monoclonal antibody will be described later.
[0034]
[Table 1]

In the following, the steps from the culture of Chlamydia pneumoniae host cells to the determination of the gene DNA sequence / amino acid sequence of the Chlamydia pneumoniae antigen polypeptide will be explained step by step.
[0035]
Example 1 Preparation of DNA encoding Chlamydia pneumoniae specific 53K antigen polypeptide
(A) Host cell (HL cell) culture
Pre-plastic culture flask (75cm ² HL cells grown on the entire bottom surface of () were washed with 5 ml of phosphate buffered saline (hereinafter referred to as PBS) magnesium-free (−) solution and PBS containing 0.1% (w / v) trypsin. Add 5 ml of the solution to the whole cell surface, discard the solution, incubate at 37 ° C. for 10 minutes, add 5 ml of Dulbecco's MEM medium containing 10% (v / v) fetal calf serum, and remove HL cells by pipetting. After exfoliation, a cell suspension was prepared.
[0036]
75cm ² When culturing in a plastic culture flask, add 1 ml of the above cell suspension and 15-20 ml of Dulbecco's MEM medium containing 10% (v / v) fetal calf serum to the culture flask, and culture in a 6-well plastic culture container. In this case, 4 ml each of a mixture of 8 ml of the cell suspension and 292 ml of Dulbecco's MEM medium containing 10% fetal bovine serum was added to each well and cultured in a 5% (v / v) carbon dioxide atmosphere.
[0037]
(B) Culture of Chlamydia pneumoniae YK41
The culture supernatant of HL cells grown in a 6-well plastic culture vessel (on the bottom) was removed with a pipette, and this was added to the Chlamydia pneumoniae strain YK41 (Kanemoto et al .: Microbiol. Immunol., Vol. 37, P.495-498). , 1993) [Liquid Chlamydia pneumoniae YK41 storage solution (hereinafter referred to as SPG) containing 75 g of sucrose, 0.52 g of monopotassium phosphate, 1.22 g of dipotassium phosphate and 0.72 g of glutamic acid per liter. 2), diluted with 12 to 24 times, treated with ultrasound for 1 minute, and centrifuged at 2,000 rpm for 3 minutes] was added to each well and centrifuged at 2,000 rpm for 1 hour. After centrifugal adsorption, Chlamydia pneumoniae suspension was removed, 4 ml of Dulbecco's MEM medium containing 1 μg / ml cycloheximide and 10% (v / v) fetal calf serum was added per well, and 5% (v / v) carbon dioxide gas atmosphere. The cells were cultured at 36 ° C. for 3 days. After culturing, the cells were detached with a sterilized silicon piece, and the cells were collected. This was centrifuged at 8,000 rpm for 30 minutes and the precipitate was resuspended in SPG and stored at -70 ° C.
[0038]
(C) Purification of the basic body of Chlamydia pneumoniae YK41
The frozen HL cell suspension of Chlamydia pneumoniae YK41 stored at -70 ° C was thawed and homogenized with a Teflon homogenizer. Centrifugation was performed at 2,500 rpm for 10 minutes, and the supernatant was collected. The precipitate was again suspended in SPG, the same operation was performed, and the supernatant was collected. The same operation was further performed twice, and the resulting supernatants were collected and combined.
[0039]
Separately, 0.03M Tris-HCl buffer (pH 7.4) containing 50% (w / v) sucrose in a centrifuge tube, then 3 volumes of urografin 76% (manufactured by Schering) and 0.03M Tris-HCl buffer A liquid mixture with 7 volumes of a liquid (pH 7.4) was layered, and the supernatant collected earlier was carefully layered thereon and centrifuged at 8,000 rpm for 1 hour. A 0.03M Tris-HCl buffer solution (pH 7.4) layer containing 50% (w / v) sucrose and a precipitate were collected, and the same volume of SPG was added to the collected solution, followed by centrifugation at 10,000 rpm for 30 minutes. . The supernatant was discarded and the precipitate was suspended in SPG. Prepare a continuous density gradient solution from 35% to 50% (volume ratio of the former to the total volume) of urografin 76% (manufactured by Schering) and 0.03M Tris-HCl buffer (pH 7.4) in the centrifuge tube. Then, the suspension was layered thereon and centrifuged at 8000 rpm for 1 hour. A yellowish cloudy band corresponding to the basic body of Chlamydia pneumoniae YK41 was recovered, diluted twice with SPG, and centrifuged at 10,000 rpm for 30 minutes. The obtained precipitate was suspended in SPG, and the protein concentration was measured (using a protein measurement kit manufactured by BioRad, with bovine serum albumin as a standard), and then stored at -70 ° C.
[0040]
(D) Preparation of genomic DNA of Chlamydia pneumoniae strain YK-41
A 300 μl suspension (protein concentration: 1.37 mg / ml) of the purified Chlamydia pneumoniae YK-41 basic body was centrifuged at 4 ° C. and 12,000 rpm for 5 minutes. To the precipitate, 500 μl of 10 mM Tris-HCl buffer solution pH 8.0 (hereinafter referred to as TE buffer solution) containing 1 mM EDTA was added and suspended. The same centrifugation was performed again, and the precipitate was suspended in 300 μl of TE buffer. 30 μl of 1% SDS aqueous solution and 30 μl of 1 mg / ml proteinase K aqueous solution were added and incubated at 56 ° C. for 30 minutes to dissolve the basic bodies. 350 μl of 0.1 M Tris-HCl buffer (pH 8.0) saturated phenol was added and mixed well with a vortex mixer, and then centrifuged at 4 ° C. and 12,000 rpm for 5 minutes to recover the aqueous layer (DNA extraction). This extraction operation was repeated once more. 2 μl of 10 mg / ml RNase solution was added and incubated at 37 ° C. for 2 hours to degrade RNA. Add 300 μl of a 25: 24: 1 (volume ratio) mixture of 0.1M Tris-HCl buffer (pH 8.0) saturated phenol, chloroform and isoamyl alcohol (hereinafter referred to as PCI), and mix well with a vortex mixer. Centrifugation was performed at 4 ° C. and 12,000 rpm for 5 minutes, and the aqueous layer was recovered. This operation was repeated a total of 5 times.
[0041]
1/10 volume of 10M ammonium acetate aqueous solution and 2 volumes of ethanol were added to the obtained liquid, and the mixture was allowed to stand for 5 minutes to precipitate DNA, followed by centrifugation at 4 ° C. and 12,000 rpm for 5 minutes. The precipitate was mixed with 600 μl of 70% ethanol aqueous solution, mixed, and washed twice by centrifugation at 4 ° C. and 12,000 rpm for 5 minutes. The precipitate was dried for 15 minutes with the lid of the centrifuge tube open, and dissolved by adding 200 μl of TE, and stored at −20 ° C.
[0042]
(E) Preparation of genomic DNA expression library
To 100 μl of genomic DNA solution, 10 μl of restriction enzyme M-buffer and 10 μl of a restriction enzyme mixture (AccI, HaeIII and 0.4 μl each of AluI diluted 1/50 and TE 20 μl) were added and reacted at 37 ° C. for 20 minutes. The reaction time of 20 minutes was a time during which DNA was decomposed into partially digested DNA having a size of 1 kbp to 7 kbp, and was tested in advance using a small amount of genomic DNA. 100 μl of PCI was added to the reaction solution, mixed well with a vortex mixer, and centrifuged at 12,000 rpm for 5 minutes at 4 ° C. to recover the aqueous layer. To this was added 10 μl of 3M sodium acetate aqueous solution and 220 μl of ethanol, and the mixture was allowed to stand at −80 ° C. for 15 minutes to precipitate partially digested DNA. Centrifugation was performed at 4 ° C. and 12,000 rpm for 5 minutes. After discarding the supernatant, 500 μl of a 70% ethanol aqueous solution was added to the precipitate, and the mixture was again centrifuged at 12,000 rpm for 5 minutes. The supernatant was discarded and the precipitate was dried under reduced pressure.
[0043]
The obtained partially digested DNA was dissolved in 20 μl of purified water, and 19 μl thereof was taken, and 14 μl of a linker (20 pmole / μl) shown in the following chemical formula 1 (10 mM ATP4. 5 μl, 50 mM MgCl ₂ , 4.5 μl of 0.2 M Tris-HCl buffer (pH 7.6, hereinafter referred to as 10-fold concentration ligation buffer) containing 50 mM dithiothreitol and 500 μg / ml bovine serum albumin, 2 μl of purified water and 1 μl of T4 ligase were added. The mixture was reacted at 16 ° C. for 4 hours to add a linker.
[Chemical 1]

[0044]
The partially digested DNA to which the linker was added was applied to a Chroma spin 6000 column using 10 mM Tris-HCl buffer containing 0.1 M NaCl and 1 mM EDTA as a mobile phase. Two drops of the eluate were collected, and a part of each fraction was analyzed by 0.8% agarose gel electrophoresis, and fractions containing DNA fragments of 1 kbp to 7 kbp were collected. To 144 μl of the obtained fraction, purified water 13 μl, 10 mM ATP 20 μl, 0.1 M MgCl ₂ 20 μl of 0.5 M Tris-HCl buffer (pH 7.6, hereinafter referred to as a 10-fold concentration phosphorylation buffer) containing 50 mM dithiothreitol, 1 mM spermidine hydrochloride and 1 mM EDTA, and 3 μl of T4 polynucleotide kinase In addition, the mixture was reacted at 37 ° C. for 30 minutes to phosphorylate the 5 ′ end of the DNA fragment. After adding 200 μl of PCI and shaking well, the mixture was centrifuged at 4 ° C. and 12,000 rpm for 5 minutes to recover the aqueous layer. Nucleotide was precipitated by adding 1 μl of 20 mg / ml aqueous glycogen solution, 20 μl of 3M aqueous sodium acetate solution and 400 μl of ethanol. Centrifugation at 4 ° C., 12,000 rpm for 10 minutes, discarding the supernatant, mixing the precipitate with 200 μl of 70% ethanol, centrifuging again, discarding the supernatant, air-drying the precipitate, and adding 1 μl of purified water to dissolve .
[0045]
1 μl of λgt11 DNA (1 μg / μl, Stratagene) previously cleaved with the restriction enzyme EcoRI, 0.5 μl of 10-fold concentration ligation buffer, 10 μm ATP 0.5 μl, T4 ligase 0. 4 μl and 2 μl of purified water were added and reacted at 4 ° C. overnight. The resulting recombinant λgt11 DNA was then packaged using a Gigapack II Gold packaging kit (Stratagene).
[0046]
(F) Preparation of Chlamydia pneumoniae specific monoclonal antibody
Culture and passage of myeloma cell lines
The myeloma cell line used for the production of the monoclonal antibody is P3 / NSI / 1-Ag4-1 (ATCC TIB-18). Cultured and passaged in RPMI 1640 medium containing 10% (v / v) fetal calf serum. Two weeks prior to cell fusion, 0.13 mM 8-azaguanine, 0.5 μg / ml MC-210 (Mycoplasma remover, Dainippon Pharmaceutical Co., Ltd.) and 10% (v / v) fetal calf serum And then cultured in a normal medium for 1 week thereafter.
[0047]
Mouse immunity
200 μl of the above suspension of the basic body having a protein concentration of 270 μg / ml was centrifuged at 12000 rpm for 10 minutes, and 200 μl of PBS was added to the precipitate and resuspended. To this, 200 μl of Freund's complete adjuvant was added to form an emulsion, and 150 μl was injected subcutaneously on the back of the mouse (this day is defined as day 0). On days 14, 34 and 49, mice were injected intraperitoneally with 100 μl of a purified elementary body suspension with a protein concentration of 270 μg / ml. Further, 50 μl of a purified basic body suspension having a protein concentration of 800 μg / ml was injected into the abdominal cavity on day 69, and 100 μl of the suspension was injected intraperitoneally on day 92, and the spleen was removed on day 95, It used for cell fusion.
[0048]
Cell fusion
Spleen cells 10 obtained from the spleen of an immunized mouse ⁸ 10 myeloma cells per cell ⁷ The pieces were placed in a round bottom glass tube, mixed well, centrifuged at 1400 rpm for 5 minutes, the supernatant was removed, and the cells were further mixed well. 0.4 ml of RPMI 1640 medium containing 30% (w / v) polyethylene glycol previously kept at 37 ° C. was added and left for 30 seconds. After centrifugation at 700 rpm for 6 minutes, add 10 ml of RPMI 1640 medium, slowly rotate the glass tube to mix well with polyethylene glycol, centrifuge at 1400 rpm for 5 minutes, remove the supernatant completely, and add 5 ml of HAT to the precipitate. Medium was added and left for 5 minutes. Further, 10-20 ml of HAT medium was added, and after standing for 30 minutes, the myeloma cell concentration was 3.3 × 10 ^Five The HAT medium was added to suspend the cells so that the amount of the solution became / ml, and two drops were dispensed into the wells of a 96-well plastic culture vessel using a Pasteur pipette. After culturing at 36 ° C. in a 5% (v / v) carbon dioxide atmosphere, 1 to 2 drops of HAT medium were added to the wells after 1, 7 and 14 days.
[0049]
Screening for antibody-producing cells
The purified Chlamydia pneumoniae YK41 elementary body was solubilized with 1% (w / v) SDS, dialyzed against 0.05M sodium bicarbonate buffer (pH 9.6) containing 0.02% sodium azide, and then protein. 50 μl of the solution diluted to a concentration of 1 to 10 μg / ml was placed in a well of a 96-well EIA plate made of vinyl chloride and allowed to stand overnight at 4 ° C. to adsorb the antigen. The supernatant was removed, 150 μl of PBS containing 0.02% (w / v) Tween 20 was added to the wells, left for 3 minutes, and then removed and washed. After further washing operation, 100 μl of PBS containing 1% (v / v) bovine serum albumin was added to the well and left standing at 4 ° C. overnight or more for blocking. After removing PBS containing bovine serum albumin and washing twice with PBS containing 0.02% (w / v) Tween 20, 50 μl of the culture supernatant of the fused cells was added to the wells and allowed to stand at room temperature for 2 hours. did. After washing three times in the same manner with PBS containing 0.02% (w / v) Tween 20, 50 μl of 25 ng / ml peroxidase-labeled goat anti-mouse IgG antibody was added to the wells and allowed to stand at room temperature for 2 hours. After washing with PBS containing 0.02% (w / v) Tween 20 three times in the same manner, 50 μl of ABTS solution (manufactured by KPL) was added to the wells and allowed to stand for 15 minutes to 1 hour at room temperature for color reaction. The absorbance at 405 nm was measured with a 96-well EIA plate photometer.
As a result, positive wells were found, and it was found that antibodies that react with the basic bodies were contained in the culture supernatant. The cells in each well were collected with a Pasteur pipette, transferred to a 24-well plastic culture container, added with 1-2 ml of HAT medium, and cultured in the same manner.
[0050]
Cloning by limiting dilution method
The cell concentration of the fused cells grown in a 24-well plastic culture vessel was measured, and each was diluted with HT medium so that the number of cells was 20 cells / ml. Separately, 4-6 week-old mouse thymocytes suspended in HT medium were placed in a 96-well plastic culture vessel at 2 × 10 ^Five 50 μl / well of the above fused cells (cell concentration: 20 cells / ml) was added to each cell, and the cells were cultured at 36 ° C. in a 5% (v / v) carbon dioxide atmosphere. 1-2 drops / well of HT medium was added after 14 days. 50 μl of the culture supernatant of the well in which cell proliferation was observed was collected, and antibody production was confirmed by the same method as described above.
Cells having only a single cell colony in the well and producing an antibody that reacts with the basic body and rapidly growing were collected from the well and subsequently grown in a 24-well plastic culture vessel. Further, the same cloning operation was repeated to finally obtain a hybridoma, AY6E2E8.
[0051]
Production of monoclonal antibodies
75 cm of hybridoma AY6E2E8 containing 20 ml of RPMI 1640 medium containing 10% (v / v) fetal calf serum ² Grow in plastic cell culture flasks and remove 16-18 ml from the culture every 3-4 days, instead use fresh 10% (v / v) fetal calf serum-containing RPMI 1640 medium to a total volume of 20 ml The subculture was continued. The cell culture solution extracted and collected was centrifuged at 1200 rpm for 5 minutes, and the supernatant (culture supernatant containing the monoclonal antibody) was collected.
In addition, 1 to 5 × 10 5 in the abdominal cavity of a Balb / c mouse that had been injected intraperitoneally with 0.5 ml of pristane two weeks ago ⁶ 1 ml of a hybridoma strain suspended in PBS so that the number of cells / ml was injected. After 3 weeks, the ascites of balb / c mice was collected and centrifuged at 1200 rpm for 5 minutes, and the supernatant (monoclonal antibody-containing ascites) was collected.
[0052]
Purification of monoclonal antibodies
The monoclonal antibody produced by the hybridoma AY6E2E8 was purified as follows. Hybridoma AY6E2E8 was injected intraperitoneally into a mouse, and 1 volume of monoclonal antibody-containing ascites was mixed with 3 volumes of PBS, centrifuged at 3000 rpm for 10 minutes, and the supernatant filtered through a filter with a pore size of 0.22 μm. Thereafter, this was purified by HPLC using a Chromato Super Protein A column (diameter: 4.6 mm × 100 mm, manufactured by NGK Co., Ltd.). The column was previously equilibrated with PBS.
After injecting 1 ml of the sample after filtration through a 0.22 μm filter onto the column, PBS was flushed at 1 ml / min for 3 minutes, and then at 5 ml / min for 4 minutes to wash the column, and then 8.77 g of NaCl in 1 L of purified water. , 16.7 g of citric acid (monohydrate) and Na ₂ HPO _Four ・ 12H ₂ The monoclonal antibody was eluted by flowing a solution of 14.72 g of O at 2 ml / min for 5 minutes. Monoclonal antibody elution fractions were collected and diluted with TTBS solution.
The basic body of Chlamydia pneumoniae was dissolved, and the peptide contained in the basic body was obtained. Western blotting was performed using this peptide and the above monoclonal antibody, and the specificity of the obtained monoclonal antibody was confirmed.
As a result, it was found that the obtained monoclonal antibody recognizes Chlamydia pneumoniae 53 KDa antigen polypeptide.
Hybridoma SCP53 and hybridoma 70 were obtained in the same manner as hybridoma AY6E2E8. As a result of examining the specificity of monoclonal antibodies produced by hybridoma SCP53 and hybridoma 70 in the same manner as described above, these monoclonal antibodies recognize 53 KDa antigen polypeptide and 73 KDa antigen polypeptide of Chlamydia pneumoniae, respectively. I understood.
Further, as a result of examining the subclasses of the monoclonal antibodies produced by the hybridoma SCP53 and the hybridoma 70 in the same manner as described above, the subclasses of these antibodies are IgG, respectively. ₁ And IgG.
[0053]
(G) Cloning of DNA encoding the antigen polypeptide
One platinum loop of E. coli Y1090r-strain is added to 10 mM MgSO _Four After inoculating 3 ml, LB containing 0.2% maltose and 50 μg / ml ampicillin (containing 5 g of NaCl, 10 g of polypeptone and 5 g of yeast extract in 1 L of water) and culturing with shaking at 37 ° C. overnight, Centrifugation was performed at 2,000 rpm for 10 minutes. 10 mM MgSO for precipitation (E. coli) _Four Add 9 ml of aqueous solution and mix. Take 0.35 ml of this Escherichia coli suspension, add 0.1-10 μl of λgt11 (DNA library) suspension to this and incubate at 37 ° C. for 20 minutes. Infected with λgt11. The λgt11-infected Escherichia coli was added to 2.5 ml of a liquid LB agar medium previously maintained at 47 ° C., and this was immediately spread on the LB agar medium. After the upper agar medium has solidified, it is cultured at 42 ° C. for 3 to 4 hours. When plaques are observed, a nitrocellulose filter (φ82 mm) immersed in 10 mM IPTG aqueous solution is placed on the upper agar medium and cultured at 37 ° C. for 12 hours. did. After three asymmetrical punctures with a black ink injection needle to mark the filter, the filter was removed from the agar medium, and 20 mM Tris-HCl buffer (pH 7.5 containing 150 mM NaCl and 0.1% Tween 20). ) (Hereinafter referred to as TTBS buffer). The agar medium was stored in a refrigerator.
[0054]
The filter is immersed in a solution containing 0.1% bovine serum albumin in 20 mM Tris-HCl buffer (pH 7.5) (hereinafter referred to as TBS buffer) containing 150 mM NaCl, and shaken at 37 ° C. for 1 hour to perform a blocking reaction. It was. Next, the filter was washed twice with TTBS buffer, immersed in TTBS solution of 5-10 μg / ml of Chlamydia pneumoniae-specific monoclonal antibody (SCP53 or AY6E2E8), and shaken at 37 ° C. for 1 hour. The filter was washed 3 times with TTBS buffer, and then shaken in a peroxidase-labeled (50 ng / ml) anti-mouse IgG antibody solution (TTBS buffer) at 37 ° C. for 1 hour. The filter was washed 3 times with TTBS buffer and 3 times with TBS buffer, and then the chromogenic substrate solution (60 ml of 30% hydrogen peroxide solution and methanol of 0.3% 4-chloro-1-naphthol in 100 ml of TBS buffer) 20 ml of the solution was added to the preparation) and left at room temperature for about 30 minutes. When the color was sufficiently developed, the filter was taken out, washed with purified water, and air-dried.
[0055]
Plaques on the agar medium corresponding to the colored spots on the filter were searched and identified, and the agar in this part was stabbed with a Pasteur pipette to collect the plaques. The collected plaque was taken up in 50 mM Tris-HCl buffer (pH 7.5) (hereinafter referred to as SM buffer) containing 0.1 M NaCl, 8 mM magnesium sulfate and 0.01% gelatin to which 1 drop of chloroform was added. The λ phage in the plaque was extracted by leaving overnight. The above procedure was repeated until all the plaques reacted with the monoclonal antibody, and the DNA encoding the antigen polypeptide was cloned.
Thus, a λ phage expressing a Chlamydia pneumoniae-specific monoclonal antibody-reactive Chlamydia pneumoniae-specific antigen polypeptide was obtained, which was designated as 53-3Sλ phage.
[0056]
(H) Culture of 53-3Sλ phage and DNA purification
Plaques were formed in the same manner as described in (G) above, and one plaque was collected, placed in 100 μl of SM buffer, and left overnight at 4 ° C. to extract λ phage. 5 to 10 μl of λ phage solution was added to 250 μl of E. coli Y1090r strain cultured overnight in LB medium, and left at 37 ° C. for 20 minutes to infect Escherichia coli with λ phage. 50 ml of LB medium containing 10 mM magnesium sulfate previously warmed to 37 ° C. was inoculated, and cultured with shaking at 37 ° C. for 5 to 7 hours until lysis of E. coli by λ phage occurred. 250 μl of chloroform was added and centrifuged at 3,000 rpm for 10 minutes to remove E. coli cell residues, and a λ phage suspension was obtained. λ phage DNA was purified using Wizard λ preps kit (Promega).
[0057]
(I) Amplification of DNA encoding Chlamydia pneumoniae antigen polypeptide
In a microtube for 600 μl, purified water 61.5 μl, 10-fold concentration reaction buffer (500 mM KCl, 15 mM MgCl ₂ Tris-HCl buffer solution containing 0.01% gelatin, pH 8.3) 10 μl, 20 mM dNTP 1 μl, 53-3Sλ phage DNA solution 0.1 μl, 20 nM λgt11 forward primer (Takara Shuzo Co., Ltd.) 1 μl, 20 nM λgt11 reverse primer (Takara Shuzo) Co.) 1 μl and AmpliTaq DNA Polymerase 0.5 μl were added, and 2 to 3 drops of mineral oil were overlaid. Incubation with a cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 73 ° C. for 2 minutes was repeated 30 times to amplify the DNA. After the reaction, 1.2% low-temperature melting agarose gel electrophoresis was performed, and the amplified DNA was excised and purified with Wizard PCR Prep kit (Promega).
[0058]
(J) DNA sequence analysis
The DNA base sequence analysis was performed with a 373A type DNA sequencer (Applied Biosystems) by using a DNA amplified by PCR as a template and performing a sequence reaction by a fluorescence-labeled terminator cycle sequencing method using Taq DNA polymerase. The obtained DNA base sequence was edited, linked, and the amino acid translation region was estimated using gene sequence analysis software “DNASIS” (Hitachi Software Engineering Co., Ltd.) to obtain the sequence of SEQ ID NO: 9.
The analysis result of the sequence of SEQ ID NO: 9 revealed that about 60% of the amino acid sequence of the 53 KDa antigen polypeptide was elucidated from the N-terminus to the C-terminus.
Since the DNA encoding the Chlamydia pneumoniae antigen polypeptide was cloned using a monoclonal antibody specific for Chlamydia pneumoniae and recognizing the 53 KDa antigen polypeptide, this DNA was clearly It encodes a peptide.
As a result of homology search of the base sequence and amino acid sequence of SEQ ID NO: 9 with the GenBank database, there was no known sequence showing high homology.
[0059]
Example 2 Production of Recombinant Vector Containing DNA Encoding Polypeptide Containing Part of Chlamydia pneumoniae Antigen Polypeptide and Transformant Containing it
As described above, it is clear that the obtained DNA encodes the 53 KDa antigen polypeptide. As a precaution, whether the obtained DNA is expressed and reacted with the antibody as described below. Examined.
Plasmid pBBK10MM was digested with restriction enzymes BamHI and XhoI, and subjected to 1.2% low-temperature melting agarose gel electrophoresis, and a DNA fragment of about 4.6 Kbp was excised and purified. 1 ng each of the synthetic DNAs of SEQ ID NO: 11 and SEQ ID NO: 12 was added to 100 ng of this DNA fragment, and these DNAs were ligated using a DNA ligation kit (Takara Shuzo). This reaction product was put into Escherichia coli HB101 strain competent cell (Takara Shuzo Co., Ltd.), a transformant was prepared, a plasmid was obtained, and this was named pADA431. This plasmid was cleaved with the restriction enzyme MunI and then treated with alkaline phosphatase to remove the 5 'phosphate group.
On the other hand, 53-3Sλ phage DNA was cleaved with the restriction enzyme EcoRI, and 100 ng of the pADA431 plasmid DNA cleaved with the restriction enzyme MunI was added to 50 ng of this DNA fragment and ligated in the same manner to prepare a transformant. A plasmid incorporating a restriction enzyme EcoRI fragment of 3Sλ phage DNA was obtained and named pCPN533α. This plasmid is a DNA of about 5.7 kbp having the base sequence of SEQ ID NO: 10, and a polypeptide containing a part of the 53K antigen polypeptide can be expressed in host E. coli. The base sequence of the DNA encoding the polypeptide containing a part of the 53K antigen polypeptide is as shown in SEQ ID NO: 4, and the amino acid sequence deduced from this base sequence is as shown in SEQ ID NO: 2. . Escherichia coli having the plasmid pCPN533α was cultured in the same manner and subjected to electrophoresis, transcription to a nitrocellulose membrane, and detection with a monoclonal antibody. As a result, a colored band corresponding to the above polypeptide was observed and the plasmid pCPN533α was present E. coli has been shown to express a 53K antigen polypeptide that can react with monoclonal antibodies that react specifically with Chlamydia pneumoniae.
[0060]
Example 3 Obtaining DNA encoding the entire 53 KDa antigen polypeptide of Chlamydia pneumoniae
Based on the nucleotide sequence of SEQ ID NO: 9, DNAs having the nucleotide sequences of SEQ ID NOs: 14 and 15 were synthesized using a DNA synthesizer.
To 10 μl of the aqueous solution of Chlamydia pneumoniae YK41 genomic DNA obtained in Example 1 (DNA content: about 1 μg), 5 μl of 10-fold concentrated K buffer, 35 μl of purified water and 5 μl of restriction enzyme HindIII (19 U / μl) were added. And kept at 37 ° C. for 3 hours.
The obtained reaction solution was extracted with phenol, ethanol was added, and the mixture was centrifuged to obtain a precipitate. To this precipitate, 5 μl of HindIII cassette DNA (20 ng / μl) in a PCR in vitro Cloning Kit (product name of Takara Shuzo Co., Ltd.) and 15 μl of ligation solution were added and incubated at 16 ° C. for 30 minutes.
The obtained reaction solution was extracted with phenol, ethanol was added, and the mixture was centrifuged to obtain a precipitate, which was dissolved in 10 μl of purified water.
To 1 μl of the obtained solution, 78.5 μl of purified water, 10 μl of 10-fold concentrated PCR buffer, 8 μl of 2.5 mM dNTP and 0.5 μl of Taq polymerase (5 U / μl) were added. 1 μl of DNA having a sequence (20 pmol / μl) and 1 μl of DNA having a nucleotide sequence of SEQ ID NO: 16 (20 pmol / μl) (enclosed as primer C1 in the above kit) were added, and 0.6 ml of these were added. Were placed in a microtube and two drops of mineral oil were overlaid, and a temperature cycle of 94 ° C. for 30 seconds, 55 ° C. for 2 minutes, and 72 ° C. for 3 minutes was repeated 30 times. The above process is called a PCR process.
In 1 μl of the reaction solution after the PCR step, 1 μl of DNA having the base sequence of SEQ ID NO: 15 (20 pmol / μl) and DNA having the base sequence of SEQ ID NO: 17 (20 pmol / μl) are used as primer DNA (in the above kit, primer C2 The PCR step was performed again using 1 μl.
The reaction solution after the second PCR step was subjected to 1.2% low-melting point agarose gel electrophoresis, and an agarose gel containing DNA having a size of about 1.4 kbp was cut out. Wizard PCR Prep kit (Promega) was used for DNA purification. That is, the buffer solution enclosed in the kit is added to the cut-out agarose gel, heated to dissolve the agarose gel, the purification resin enclosed in the kit is added to adsorb the DNA to the resin, and centrifuged. Thus, the purification resin was obtained as a precipitate. The precipitate was washed with propanol and centrifuged again to obtain a precipitate. Purified water was added to the precipitate, DNA was eluted from the purification resin, and centrifuged to obtain a supernatant (aqueous DNA solution). The above process is called a DNA purification process.
The obtained DNA aqueous solution is used to perform a sequencing reaction by a fluorescence labeled terminator cycle sequencing method using Taq DNA polymerase using the contained DNA as a template, and the base sequence of the DNA is analyzed with a 373A type DNA sequencer (Applied Biosystems). did. Using the DNA sequence analysis software “DNASIS” (Hitachi Software Engineering), the obtained DNA base sequence was edited, linked, and the amino acid translation region was estimated. The above process is called a base sequence analysis process.
As a result of analyzing the base sequence of the obtained DNA, this DNA had a base sequence of about 50 bp on the 3 ′ end side in the DNA encoding the Chlamydia pneumoniae antigen polypeptide obtained in Example 1. Furthermore, it was found that a coding region of about 0.7 kb containing a termination codon was present downstream of the base sequence. Based on the base sequence of SEQ ID NO: 9, as a primer corresponding to the upstream portion of the DNA encoding the Chlamydia pneumoniae antigen polypeptide, the DNA having the base sequence of SEQ ID NO: 18 is also about 0.7 kb as described above. Using the DNA synthesizer, each of the DNA having the nucleotide sequence of SEQ ID NO: 19 as a primer corresponding to the downstream portion of the DNA encoding the Chlamydia pneumoniae antigen polypeptide based on the nucleotide sequence comprising the coding region of And synthesized.
Using 1 μl of an aqueous solution of Chlamydia pneumoniae YK41 genomic DNA obtained in Example 1, 1 μl of DNA having the nucleotide sequence of SEQ ID NO: 18 (20 pmol / μl) as the primer DNA and DNA having the nucleotide sequence of SEQ ID NO: 19 (20 pmol) The PCR step was performed using 1 μl.
Using the reaction solution after the third PCR step, the DNA purification step was performed to obtain about 1.5 kbp of DNA.
Using the obtained aqueous DNA solution, the above base sequence analysis step was performed.
As a result of analyzing the base sequence of the obtained DNA, it was found that this DNA has the base sequence of SEQ ID NO: 4 and encodes the amino acid sequence of SEQ ID NO: 1. Further, genome walking was performed using the plasmid pCPN533α and the above-mentioned λgt11 DNA library to obtain DNA encoding the entire 53 KDa antigen polypeptide of Chlamydia pneumoniae.
[0061]
Example 4 Production of a recombinant vector containing DNA encoding the entire 53 KDa antigen polypeptide of Chlamydia pneumoniae and production of a transformant containing the same
Using a DNA encoding the entire Chlamydia pneumoniae 53 KDa antigen polypeptide, a recombinant vector containing a DNA encoding the entire Chlamydia pneumoniae 53 KDa antigen polypeptide and a transformant containing the same were prepared in the same manner as in Example 2. To do.
[0062]
Example 5 Preparation of DNA encoding Chlamydia pneumoniae 73K antigen polypeptide
Monoclonal antibody 70 was used instead of monoclonal antibody SCP53 or AY6E2E8, and the same procedure as in Example 1 was used. Clone 70-2Sλ phage was obtained, from which the sequence of SEQ ID NO: 13 was obtained.
The analysis result of the sequence of SEQ ID NO: 13 revealed that about 90% of the amino acid sequence of Chlamydia pneumoniae 73K antigen protein was elucidated from the N-terminus to the C-terminus.
As a result of homology search of the nucleotide sequence and amino acid sequence of SEQ ID NO: 13 in the GenBank database, it was found that the gene nucleotide sequence isolated from Chlamydia trachomatis (LMSardinia et al: J. Bacteriol., Vol.171, 335- 341 (1989)).
[0063]
Example 6 Production of anti-Chlamydia pneumoniae antibody using Chlamydia pneumoniae antigen polypeptide as an antigen
(A) Culture and passage of myeloma cell line
The myeloma cell line is subcultured by culturing P3X63Ag8.653 (ATCC CRL-1580) in RPMI 1640 medium containing 10% (v / v) fetal calf serum. Two weeks prior to cell fusion, 0.13 mM 8-azaguanine, 0.5 μg / ml MC-210 (Mycoplasma remover, Dainippon Pharmaceutical Co., Ltd.) and 10% (v / v) fetal calf serum Incubate for 1 week in RPMI 1640 medium containing, and in a normal medium for the following 1 week.
[0064]
(B) Mouse immunity
200 μl of the suspension of the above antigen polypeptide having a protein concentration of 270 μg / ml is centrifuged at 12000 rpm for 10 minutes, and 200 μl of PBS is added to the precipitate and resuspended. 200 μl of Freund's complete adjuvant is added to this to make an emulsion, and 150 μl is injected subcutaneously on the back of the mouse (this day is designated as day 0). On days 14, 34 and 49, 100 μl of the suspension of the above antigen polypeptide having a protein concentration of 270 μg / ml was injected into the abdominal cavity of the mouse, and on day 69, the protein concentration was 800 μg. 50 μl / ml of the above-mentioned antigen polypeptide suspension and 100 μl of the suspension on day 92 are injected into the abdominal cavity of the mouse. On day 95, the spleen is removed and subjected to cell fusion.
[0065]
(C) Cell fusion
Spleen cells 10 obtained from the spleen ⁸ 10 myeloma cells per cell ⁷ Take the pieces into a round bottom glass tube, mix well, centrifuge at 1400 rpm for 5 minutes, remove the supernatant and mix the cells better. Add 0.4 ml of RPMI 1640 medium containing 30% (w / v) polyethylene glycol, which has been kept at 37 ° C., and let stand for 30 seconds. After centrifugation at 700 rpm for 6 minutes, add 10 ml of RPMI 1640 medium, slowly rotate the glass tube to mix well with polyethylene glycol, centrifuge at 1400 rpm for 5 minutes, completely remove the supernatant, and add 5 ml of HAT to the precipitate. Add medium and leave for 5 minutes. Further, add 10-20 ml of HAT medium and let stand for 30 minutes. The myeloma cell concentration is 3.3 × 10. ^Five Suspend cells by adding HAT medium to make / ml, and dispense 2 drops into wells of a 96-well plastic culture vessel using a Pasteur pipette. Incubate at 36 ° C. in a 5% (v / v) carbon dioxide atmosphere, and add 1 to 2 drops of HAT medium to the wells after 1, 7 and 14 days.
[0066]
(D) Screening of antibody producing cells
The antigen polypeptide is suspended in 0.05M sodium bicarbonate buffer (pH 9.6) containing 0.02% (w / v) sodium azide so that the protein concentration becomes 1 to 10 μg / ml, and 0.02% Dialyzed against 0.05 M sodium bicarbonate buffer (pH 9.6) containing sodium fluoride, and then diluted to a protein concentration of 1 to 10 μg / ml was added to the wells of a 96-well EIA plate made of vinyl chloride. Take 50 μl and leave overnight at 4 ° C. to adsorb the antigen. The supernatant is removed, 150 μl of PBS containing 0.02% (w / v) Tween 20 is added to the wells, left for 3 minutes, and then removed and washed. After further washing operation, 100 μl of PBS containing 1% (v / v) bovine serum albumin is added to the well and left standing at 4 ° C. overnight or more for blocking. After removing PBS containing bovine serum albumin and washing twice with PBS containing 0.02% (w / v) Tween 20, 50 μl of the culture supernatant of the fused cells was added to the wells and allowed to stand at room temperature for 2 hours. To do. After washing three times in the same manner with PBS containing 0.02% (w / v) Tween 20, 50 μl of 25 ng / ml peroxidase-labeled goat anti-mouse IgG antibody is added to the wells and left at room temperature for 2 hours. After washing three times in the same manner with PBS containing 0.02% (w / v) Tween 20, 50 μl of ABTS solution (manufactured by KPL) was added to the wells and allowed to stand at room temperature for 15 minutes to 1 hour for color reaction. Absorbance at 405 nm is measured with a photometer for well EIA plates. Cells in positive wells are collected with a Pasteur pipette, transferred to a 24-well plastic culture vessel, added with 1-2 ml of HAT medium, and cultured in the same manner.
[0067]
(E) Cloning by limiting dilution method
The cell concentration of two strains of fused cells grown in a 24-well plastic culture vessel is measured, and each cell is diluted with HT medium so that the number of cells becomes 20 cells / ml. Separately, 4-6 week-old mouse thymocytes suspended in HT medium were placed in a 96-well plastic culture container at 1-2 × 10. ^Five 50 μl / well of the above fused cells (cell concentration: 20 cells / ml) was added to each cell, and the cells were cultured at 36 ° C. in a 5% (v / v) carbon dioxide atmosphere. Add 1-2 drops / well of HT medium after 14 days. 50 μl of the culture supernatant of a well in which cell growth was observed is collected, and antibody production is confirmed by the same method as in “Screening of antibody-producing cells” in (D) above.
Cells that produce antibodies that have only a single cell colony in the well and react with the elementary bodies and that grow rapidly are collected from the well and subsequently grown in a 24-well plastic culture vessel. Further, the same cloning operation is repeated to obtain a hybridoma producing an anti-Chlamydia pneumoniae antibody. This is cultured, and an anti-Chlamydia pneumoniae antibody is produced from the culture supernatant.
[0068]
【The invention's effect】
The antigen polypeptide of claim 1 can be used for antibody testing of Chlamydia pneumoniae.
The antigen polypeptide according to claim 2 exhibits the effect of the antigen polypeptide according to claim 1, and further, since the length of the amino acid sequence is short, the number of antigen peptides that can be immobilized on a carrier or the like can be increased. Thereby, it can be used for the production of a highly sensitive diagnostic agent.
The antigen polypeptide according to claim 3 exhibits the effects of the antigen polypeptide according to claim 1, and can form a structure that is difficult to be degraded by a proteolytic enzyme, so that it is excellent in stability as an antigen.
The antigen polypeptide according to claim 4 exhibits the effect of the antigen polypeptide according to claim 1, and further can be immobilized on a carrier or the like using an amino acid or a 2-1000 amino acid sequence. Sexual decline or loss is unlikely to occur.
The antigen polypeptide according to claim 5 exhibits the effect of the antigen polypeptide according to claim 1, and further has the whole antigen polypeptide specific to Chlamydia pneumoniae, so that the accuracy of antibody test and Chlamydia pneumoniae infection can be improved. It is extremely suitable for simple diagnosis.
The antigen polypeptide according to claim 6 exhibits the effect of the antigen polypeptide according to claim 1, and further has an antigen portion specific to Chlamydia pneumoniae, so that it can be used for antibody tests and accurate diagnosis of Chlamydia pneumoniae infection. Very appropriate.
The antigen polypeptide according to claim 7 has the effect of the antigen polypeptide according to claim 1 and further has an antigen portion specific to Chlamydia pneumoniae, so that it can be used for antibody tests and accurate diagnosis of Chlamydia pneumoniae infection. Very appropriate.
The DNA according to claim 8 can be used for the production of an antigen polypeptide suitable for testing for Chlamydia pneumoniae antibodies, diagnosis of Chlamydia pneumoniae infection, and the like.
The DNA according to claim 9 has the effect of the DNA according to claim 8, and further, since the antigen polypeptide encoded by this DNA has the whole antigen polypeptide specific to Chlamydia pneumoniae, It can be used to produce an antigen polypeptide suitable for pneumoniae specific antibody testing and the like.
The DNA according to claim 10 exhibits the effect of the DNA according to claim 8, and further, since the antigen polypeptide encoded by this DNA has an antigen portion specific to Chlamydia pneumoniae, the specificity of Chlamydia pneumoniae is unique. It can be used for the production of an antigen polypeptide that is suitable for a typical antibody test or the like.
The DNA according to claim 11 exhibits the effect of the DNA according to claim 8, and the antigen polypeptide encoded by this DNA has an antigen portion specific to Chlamydia pneumoniae, so that the specificity of Chlamydia pneumoniae is unique. It can be used for the production of an antigen polypeptide that is suitable for a typical antibody test or the like.
The recombinant vector according to claim 12 can be used for antibody testing of Chlamydia pneumoniae and production of an antigen polypeptide suitable for diagnosis of Chlamydia pneumoniae infection.
The recombinant vector according to claim 13 exhibits the effect of the recombinant vector according to claim 12, and can express a polypeptide having an antigen portion specific to Chlamydia pneumoniae. It can be used for the production of an antigen polypeptide that is extremely suitable for specific antibody testing and the like.
The transformant according to claim 14 can be used for the production of an antigen polypeptide suitable for testing for a specific antibody of Chlamydia pneumoniae.
The method for producing an anti-Chlamydia pneumoniae antibody according to claim 15 can be used for producing a diagnostic agent for Chlamydia pneumoniae infection.
[0069]
[Sequence Listing]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

Claims (20)

A polypeptide comprising SEQ ID NO: 1 comprising at least 20 consecutive amino acid sequences that retain reactivity with the antibody against the polypeptide of SEQ ID NO: 1, and reacting with the antibody against the polypeptide of SEQ ID NO: 1 Retained Chlamydia pneumoniae antigen polypeptide. SEQ ID NO: 1 in a polypeptide in which one amino acid in the polypeptide of SEQ ID NO: 1 is substituted with another amino acid or one amino acid is inserted in the polypeptide of SEQ ID NO: 1 An antigenic polypeptide of Chlamydia pneumoniae comprising a sequence of at least 20 consecutive amino acids that retains reactivity with an antibody against the polypeptide of claim 1, and retains reactivity with the antibody against the polypeptide of SEQ ID NO: 1. Comprising at least 20 contiguous amino acid sequences in a polypeptide wherein one glycine in the polypeptide of SEQ ID NO: 1 is substituted with alanine and / or one alanine is substituted with glycine; An antigenic polypeptide of Chlamydia pneumoniae that retains reactivity with an antibody against the polypeptide of SEQ ID NO: 1. A polypeptide in which an amino acid or a peptide is bound to at least 20 consecutive amino acid sequences that retain reactivity with an antibody against the polypeptide of SEQ ID NO: 1 among the polypeptides of SEQ ID NO: 1, and SEQ ID NO: 1 An antigenic polypeptide of Chlamydia pneumoniae that retains reactivity with an antibody against the polypeptide.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 1.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 2.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 5.   DNA encoding the polypeptide according to any one of claims 1 to 7, or DNA complementary thereto.   DNA consisting of the base sequence of SEQ ID NO: 3 or DNA complementary thereto.   DNA consisting of the base sequence of SEQ ID NO: 4 or DNA complementary thereto.   DNA consisting of the base sequence of SEQ ID NO: 7 or DNA complementary thereto.   DNA consisting of the base sequence of SEQ ID NO: 9 or DNA complementary thereto.   DNA consisting of the nucleotide sequence of SEQ ID NO: 14 or DNA complementary thereto.   DNA consisting of the base sequence of SEQ ID NO: 15 or DNA complementary thereto.   DNA consisting of at least bases 1 to 18 in the base sequence of SEQ ID NO: 3 or DNA complementary thereto.   DNA consisting of at least nucleotides 1451 to 1464 in the nucleotide sequence of SEQ ID NO: 3 or DNA complementary thereto.   A recombinant vector comprising the DNA according to any one of claims 8 to 12.   The recombinant vector according to claim 17, which is a pCPN533α plasmid having the base sequence of SEQ ID NO: 10.   A transformant comprising the recombinant vector according to claim 17 or 18.   A method for producing an anti-Chlamydia pneumoniae antibody, wherein the antigen polypeptide according to any one of claims 1 to 7 is used as an antigen.