JP3692599B2 - Anti-Chlamydia pneumoniae antibody assay and reagents and diagnostic agent for Chlamydia pneumoniae infection - Google Patents

Description

【００５１】
モノクローナル抗体の作製方法については後述する。
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配列とアミノ酸配列の決定まで、順を追って説明する。
【００５２】
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。この細胞浮遊液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６ウェルプラスチック製培養容器の各ウェルに加え、５％（ｖ／ｖ）炭酸ガス雰囲気下で培養した。
【００５３】
（Ｂ）クラミジア・ニューモニエＹＫ−４１の培養
クラミジアとして、クラミジア・ニューモニエＹＫ−４１株（金本ら：Microbiol.Immunol.,Vol.37,P.495-498,1993）を用いた。
取得したＨＬ細胞に予めクラミジア・ニューモニエＹＫ−４１株を感染させておき、この細胞をＳＰＧ液に懸濁し、ポリプロピレン製遠心チューブに入れ、これに１秒間隔で３０回超音波を照射し、遠心チューブを１，５００×ｇで３分間遠心分離し、上清を取得し、クラミジア・ニューモニエ浮遊液とした。
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・ニューモニエ浮遊液を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。
【００５４】
（Ｃ）クラミジア・ニューモニエＹＫ−４１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ＹＫ−４１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。
【００５５】
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液を加え、１０，０００rpmで３０分間遠心分離した。上清を捨て、沈殿をＳＰＧ液に懸濁した。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液で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液に懸濁し、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。
【００５６】
（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。
【００５７】
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を析出させた後、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。
【００５８】
（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。
【００５９】
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】

【００６０】
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。
【００６１】
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。
【００６２】
（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。
【００６３】
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。
【００６４】
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。
【００６５】
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１μg/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。
【００６６】
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。
【００６７】
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。
【００６８】
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、１×１０⁶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。
【００６９】
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。
【００７０】
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を取得した。上記の方法と同様にしてハイブリドーマＳＣＰ５３が産生するモノクローナル抗体の特異性を調べた結果、このモノクローナル抗体は、クラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチドを認識することがわかった。
また、ハイブリドーマ ＡＹ６Ｅ２Ｅ８及びハイブリドーマＳＣＰ５３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、ＩｇＧ₂b及びＩｇＧ₁であった。
【００７１】
（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後、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０．１μｌ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。
【００７２】
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、５μg/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（ＡＹ６Ｅ２Ｅ８又はＳＣＰ５３）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。
【００７３】
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。
【００７４】
（Ｈ）５３−３Ｓλファージの培養とＤＮＡ精製
前記（Ｆ）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５μｌ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、Wizardλ preps キット（プロメガ社、商品名）を用いて精製した。
【００７５】
（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)製、商品名）１μｌ、２０ｎＭ λgt11 reverse primer（宝酒造 (株)製、商品名）１μｌ、AmpliTaq DNA Polymerase（宝酒造(株)製、商品名）０．５μｌを入れ、ミネラルオイルを２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（プロメガ社製、商品名）で精製した。
【００７６】
（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製、商品名）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(株)製、商品名）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７の配列を得た。
配列番号７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。
【００７７】
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７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。
【００７８】
実施例２ ＤＨＦＲとクラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチドの一部を含む融合タンパク質をコードする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
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。
一方、５３−３ＳλファージＤＮＡを制限酵素EcoRIで切断し、同様に約１．０KbpのＤＮＡ断片を精製した後、さらに制限酵素AvaIIで切断し、同様に約０．８KbpのＤＮＡ断片を精製した。上記の約４．６KbpのＤＮＡ断片１００ngに上記の約０．８KbpのＤＮＡ断片１００ng、配列番号８〜１１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た。
【００７９】
この形質転換体を、５０mg/Lのアンピシリンを含むＬＢ寒天培地に塗布し、３７℃で２４時間培養した。得られた大腸菌のコロニーを５０mg/Lのアンピシリンを含むＬＢ培地３mlに接種し、３７℃で一晩振とう培養した。アルカリ溶解法でプラスミドベクターを分離し、制限酵素NruIで切断し、０．８％アガロースゲル電気泳動で分析し、６１６bpと４８２２bpのＤＮＡ断片が生じている組換えプラスミドベクターをもつ大腸菌を選択した。得られた組換えプラスミドベクターをｐＣＰＮ５３３Ｔと名付けた。このプラスミドベクターは、配列番号１２の塩基配列を有する約５．４ｋｂｐのＤＮＡであり、ＤＨＦＲのＣ末端にクラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチドの一部を含むポリペプチドを連結した融合タンパク質を発現させるものである。この融合タンパク質をコードするＤＮＡの塩基配列は配列番号６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４のようになっていた。
【００８０】
実施例３ ＤＨＦＲとクラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチドの一部を含むポリペプチドの融合タンパク質の確認
プラスミドｐＣＰＮ５３３Ｔを保持する大腸菌ＨＢ１０１株１白金耳を５０mg/lのアンピシリンを含むＬＢ培地３mlに接種し、３７℃で一晩振とう培養した。この大腸菌を含む培地１０μｌに１０μｌのローディング緩衝液（０．０１％ブロモフェノールブルー、１０％メルカプトエタノール、２０％グリセロール、５％ＳＤＳを含む０．１５６Ｍトリス塩酸緩衝液、pH６．８）を加え、８０℃で５分間加熱した後、反応液を５−２０％ポリアクリルアミドグラジエントゲル電気泳動にかけた。セミドライブロッティング装置の陽極板上に、１０％メタノール、０．０５％ドデシル硫酸ナトリウムを含む０．３Ｍトリス水溶液で湿らせたろ紙１枚、１０％メタノール、０．０５％ドデシル硫酸ナトリウムを含む２５ｍＭトリス水溶液で湿らせたろ紙１枚、１０％メタノール、０．０５％ドデシル硫酸ナトリウム、４０ｍＭアミノカプロン酸を含む２５ｍＭトリス水溶液で湿らせたニトロセルロース膜１枚、上記電気泳動の終了したポリアクリルアミドゲル、４０ｍＭアミノカプロン酸を含む２５ｍＭトリス水溶液で湿らせたろ紙２枚をこの順序で重ね、陰極板をセットして２．５mA／cm²の電流密度で1時間電流を流し、ポリアクリルアミドゲル中のタンパク質をニトロセルロース膜に転写した。このニトロセルロース膜を０．１％牛血清アルブミンを含むＴＢＳ緩衝液に入れ、室温で１時間以上放置し、ブロッキングした。ニトロセルロース膜をＴＴＢＳ緩衝液で２回洗浄した後、ハイブリドーマＡＹ６Ｅ２Ｅ８が生産するモノクローナル抗体溶液（１０μg/ml ＴＴＢＳ緩衝液中）中で３７℃、１時間振とうした。ニトロセルロース膜をＴＴＢＳ緩衝液で３回洗浄した後、パーオキシダーゼ標識した抗マウスＩｇＧ抗体溶液（５０ng/ml ＴＴＢＳ緩衝液中）中で３７℃１時間振とうした。ニトロセルロース膜をＴＴＢＳ緩衝液で3回洗浄した後、発色基質液（１００mlのTBS緩衝液に６０μｌの３０％過酸化水素水溶液と２０mlの４−クロロ−１−ナフトール メタノール溶液を混合する）に入れ、室温で３０分間反応させた。ニトロセルロース膜を取り出し、精製水で洗浄した後風乾した。この結果、融合タンパク質の大きさに相当する位置に発色したバンドが観察され、プラスミドｐＣＰＮ５３３Ｔをもつ大腸菌が、クラミジア・ニューモニエ特異的に反応するモノクローナル抗体と反応することのできる５３ＫＤａ抗原を発現していることが示された。
【００８１】
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の取得
配列番号７の塩基配列を元に、配列番号１３及び１４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、商品名)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。
【００８２】
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１３の塩基配列を有するＤＮＡ（２０pmol/μl）１μｌ及び配列番号１５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。
【００８３】
ＰＣＲ工程後の反応液１μｌ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（プロメガ社製、商品名）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。
【００８４】
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製、商品名）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(株)製、商品名）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。
【００８５】
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７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１７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１８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。
【００８６】
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１７の塩基配列を有するＤＮＡ（２０pmol/μl）１μｌ及び配列番号１８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５の４８４〜１９４７番目の塩基配列を有しており、配列番号３の１６２〜６４９番目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ｐＣＰＮ５３３Ｔ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。
【００８７】
実施例５ ＤＨＦＲとクラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチド全体の融合タンパク質をコードする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
プラスミドｐＢＢＫ１０ＭＭと前記クラミジア・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ＤＨＦＲとクラミジア・ニューモニエの５３ＫＤａ抗原ポリペプチド全体の融合タンパク質をコードする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。この融合タンパク質をコードするＤＮＡの塩基配列は配列番号５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３のようになっている。
【００８８】
実施例６ 融合タンパク質を抗原として用いる抗クラミジア・ニューモニエ抗体の測定
融合タンパク質を抗原として用いる抗クラミジア・ニューモニエ抗体の測定は、以下のようにして行うことができる。
融合タンパク質として、配列番号３のアミノ酸配列からなるポリペプチドを用い、これをＰＢＳに溶解し、マイクロタイタープレートのウェル（くぼみ）に注ぎ、４℃で一晩以上放置し、これにより融合タンパク質をマイクロタイタープレートの固定（物理吸着）させる。続いて、牛血清アルブミンを含むＰＢＳ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、ウェルに患者の血清を加え、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、ウェルにパーオキシダーゼ標識化マウス抗ヒトＩｇＧ抗体を加え、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。
【００８９】
【発明の効果】
請求項１記載の抗クラミジア・ニューモニエ抗体の測定法は、クラミジア・ニューモニエに対する抗体を特異的に測定することができ、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に好適である。
請求項２記載の抗クラミジア・ニューモニエ抗体の測定法は、請求項１記載の測定法の効果を奏し、さらに、抗原として用いられる融合タンパク質の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、高感度である。
請求項３記載の抗クラミジア・ニューモニエ抗体の測定法は、請求項１記載の測定法の効果を奏し、さらに、抗原として用いられる融合タンパク質が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安定性に優れており、測定結果の信頼性が高い。
【００９０】
請求項４記載の抗クラミジア・ニューモニエ抗体の測定法は、請求項１記載の測定法の効果を奏し、さらに、抗原として用いられる融合タンパク質がクラミジア・ニューモニエに特異的な抗原ポリペプチド全体を有するので、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５記載の抗クラミジア・ニューモニエ抗体の測定法は、請求項１記載の測定法の効果を奏し、さらに、抗原として用いられる融合タンパク質がクラミジア・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。
【００９１】
請求項６記載の抗クラミジア・ニューモニエ抗体の測定用試薬は、クラミジア・ニューモニエに対する抗体を特異的に測定することができ、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に好適である。
請求項７記載の抗クラミジア・ニューモニエ抗体の測定用試薬は、請求項６記載の測定用試薬の効果を奏し、さらに、抗原として用いられる融合タンパク質の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、高感度である。
請求項８記載の抗クラミジア・ニューモニエ抗体の測定用試薬は、請求項６記載の測定用試薬の効果を奏し、さらに、抗原として用いられる融合タンパク質が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安定性に優れており、測定結果の信頼性が高い。
【００９２】
請求項９記載の抗クラミジア・ニューモニエ抗体の測定用試薬は、請求項６記載の測定用試薬の効果を奏し、さらに、抗原として用いられる融合タンパク質がクラミジア・ニューモニエに特異的な抗原ポリペプチド全体を有するので、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１０記載の抗クラミジア・ニューモニエ抗体の測定用試薬は、請求項６記載の測定用試薬の効果を奏し、さらに、抗原として用いられる融合タンパク質がクラミジア・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。
【００９３】
請求項１１記載のクラミジア・ニューモニエ感染の診断薬は、クラミジア・ニューモニエに対する抗体を特異的に測定することができ、クラミジア・ニューモニエ感染の診断に好適である。
請求項１２記載のクラミジア・ニューモニエ感染の診断薬は、請求項１１記載の診断薬の効果を奏し、さらに、抗原として用いられる融合タンパク質の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、高感度である。
請求項１３記載のクラミジア・ニューモニエ感染の診断薬は、請求項１１記載の診断薬の効果を奏し、さらに、抗原として用いられる融合タンパク質が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安定性に優れており、診断結果の信頼性が高い。
【００９４】
請求項１４記載のクラミジア・ニューモニエ感染の診断薬は、請求項１１記載の診断薬の効果を奏し、さらに、抗原として用いられる融合タンパク質がクラミジア・ニューモニエに特異的な抗原ポリペプチド全体を有するので、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１５記載のクラミジア・ニューモニエ感染の診断薬は、請求項１１記載の診断薬の効果を奏し、さらに、抗原として用いられる融合タンパク質がクラミジア・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。
【００９５】
【配列表】
配列番号：1
配列の長さ：160
配列の型：アミノ酸
配列の種類：ペプチド

【００９６】
配列番号：2
配列の長さ：488
配列の型：アミノ酸
配列の種類：ペプチド

【００９７】
配列番号：3
配列の長さ：649
配列の型：アミノ酸
配列の種類：ペプチド

【００９８】
配列番号：4
配列の長さ：432
配列の型：アミノ酸
配列の種類：ペプチド

【００９９】
配列番号：5
配列の長さ：1947
配列の型：核酸
鎖の数：二本鎖
配列の種類：他の核酸 合成ＤＮＡ

【０１００】
配列番号：6
配列の長さ：1296
配列の型：核酸
鎖の数：二本鎖
配列の種類：他の核酸 合成ＤＮＡ

【０１０１】
配列番号：7
配列の長さ：1048
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
起源
生物名：C. ニューモニエ
株名：YK-41
直接の起源
クローン：53-3S
配列の特徴
特徴を表す記号：CDS
存在位置：236..1012
特徴を決定した方法：P

【０１０２】
配列番号：8
配列の長さ：29
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
GATCCTGATG TCTATTTCAT CTTCTTCAG 29
【０１０３】
配列番号：9
配列の長さ：28
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
GTCCTGAAGA AGATGAAATA GACATCAG 28
【０１０４】
配列番号：10
配列の長さ：30
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
AATTGCCATG GGGGCCCTTA ATTAATTAAC 30
【０１０５】
配列番号：11
配列の長さ：30
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
TCGAGTTAAT TAATTAAGGG CCCCCATGGC 30
【０１０６】
配列番号：12
配列の長さ：5438
配列の型：核酸
鎖の数：二本鎖
配列の種類：他の核酸 プラスミド
配列
ATCGATGTTA ACAGATCTAA GCTTAACTAA CTAACTCCGG AAAAGGAGGA ACTTCCATGA 60
TCAGTCTGAT TGCGGCGTTA GCGGTAGATC GCGTTATCGG CATGGAAAAC GCCATGCCGT 120
GGAACCTGCC TGCCGATCTC GCCTGGTTTA AACGCAACAC CTTAAATAAA CCCGTGATTA 180
TGGGCCGCCA TACCTGGGAA TCAATCGGTC GTCCGTTGCC AGGACGCAAA AATATTATCC 240
TCAGCAGTCA ACCGGGTACG GACGATCGCG TAACGTGGGT GAAGTCGGTG GATGAAGCCA 300
TCGCGGCGTG TGGTGACGTA CCAGAAATCA TGGTGATTGG CGGCGGTCGC GTTTATGAAC 360
AGTTCTTGCC AAAAGCGCAA AAACTGTATC TGACGCATAT CGACGCAGAA GTGGAAGGCG 420
ACACCCATTT CCCGGATTAC GAGCCGGATG ACTGGGAATC GGTATTCAGC GAATTCCACG 480
ATGCTGATGC GCAGAACTCT CACAGCTATG AGTTCGAAAT TCTGGAGCGG CGGATCCTGA 540
TGTCTATTTC ATCTTCTTCA GGACCTGACA ATCAAAAAAA TATCATGTCT CAAGTTCTGA 600
CATCGACACC CCAGGGCGTG CCCCAACAAG ATAAGCTGTC TGGCAACGAA ACGAAGCAAA 660
TACAGCAAAC ACGTCAGGGT AAAAACACTG AGATGGAAAG CGATGCCACT ATTGCTGGTG 720
CTTCTGGAAA AGACAAAACT TCCTCGACTA CAAAAACAGA AACAGCTCCA CAACAGGGAG 780
TTGCTGCTGG GAAAGAATCC TCAGAAAGTC AAAAGGCAGG TGCTGATACT GGAGTATCAG 840
GAGCGGCTGC TACTACAGCA TCAAATACTG CAACAAAAAT TGCTATGCAG ACCTCTATTG 900
AAGAGGCGAG CAAAAGTATG GAGTCTACCT TAGAGTCACT TCAAAGCCTC AGTGCCGCGC 960
AAATGAAAGA AGTCGAAGCG GTTGTTGTTG CTGCCCTCTC AGGGAAAAGT TCGGGTTCCG 1020
CAAAATTGGA AACACCTGAG CTCCCCAAGC CCGGGGTGAC ACCAAGATCA GAGGTTATCG 1080
AAATCGGACT CGCGCTTGCT AAAGCAATTC AGACATTGGG AGAAGCCACA AAATCTGCCT 1140
TATCTAACTA TGCAAGTACA CAAGCACAAG CAGACCAAAC AAATAAACTA GGTCTAGAAA 1200
AGCAAGCGAT AAAAATCGAT AAAGAACGAG AAGAATACCA AGAGATGAAG GCTGCCGAAC 1260
AGAAGTCTAA AGATCTCGAA GGAACAATGG ATACTGTCAA TACTGTGATG ATCGCGAAGG 1320
GGTTCGAATT GCCATGGGGG CCCTTAATTA ATTAACTCGA GAGATCCAGA TCTAATCGAT 1380
GATCCTCTAC GCCGGACGCA TCGTGGCCGG CATCACCGGC GCCACAGGTG CGGTTGCTGG 1440
CGCCTATATC GCCGACATCA CCGATGGGGA AGATCGGGCT CGCCACTTCG GGCTCATGAG 1500
CGCTTGTTTC GGCGTGGGTA TGGTGGCAGG CCCGTGGCCG GGGGACTGTT GGGCGCCATC 1560
TCCTTGCATG CACCATTCCT TGCGGCGGCG GTGCTCAACG GCCTCAACCT ACTACTGGGC 1620
TGCTTCCTAA TGCAGGAGTC GCATAAGGGA GAGCGTCGAC CGATGCCCTT GAGAGCCTTC 1680
AACCCAGTCA GCTCCTTCCG GTGGGCGCGG GGCATGACTA TCGTCGCCGC ACTTATGACT 1740
GTCTTCTTTA TCATGCAACT CGTAGGACAG GTGCCGGCAG CGCTCTGGGT CATTTTCGGC 1800
GAGGACCGCT TTCGCTGGAG CGCGACGATG ATCGGCCTGT CGCTTGCGGT ATTCGGAATC 1860
TTGCACGCCC TCGCTCAAGC CTTCGTCACT GGTCCCGCCA CCAAACGTTT CGGCGAGAAG 1920
CAGGCCATTA TCGCCGGCAT GGCGGCCGAC GCGCTGGGCT ACGTCTTGCT GGCGTTCGCG 1980
ACGCGAGGCT GGATGGCCTT CCCCATTATG ATTCTTCTCG CTTCCGGCGG CATCGGGATG 2040
CCCGCGTTGC AGGCCATGCT GTCCAGGCAG GTAGATGACG ACCATCAGGG ACAGCTTCAA 2100
GGATCGCTCG CGGCTCTTAC CAGCCTAACT TCGATCACTG GACCGCTGAT CGTCACGGCG 2160
ATTTATGCCG CCTCGGCGAG CACATGGAAC GGGTTGGCAT GGATTGTAGG CGCCGCCCTA 2220
TACCTTGTCT GCCTCCCCGC GTTGCGTCGC GGTGCATGGA GCCGGGCCAC CTCGACCTGA 2280
ATGGAAGCCG GCGGCACCTC GCTAACGGAT TCACCACTCC AAGAATTGGA GCCAATCAAT 2340
TCTTGCGGAG AACTGTGAAT GCGCAAACCA ACCCTTGGCA GAACATATCC ATCGCGTCCG 2400
CCATCTCCAG CAGCCGCACG CGGCGCATCT CGGGCAGCGT TGGGTCCTGG CCACGGGTGC 2460
GCATGATCGT GCTCCTGTCG TTGAGGACCC GGCTAGGCTG GCGGGGTTGC CTTACTGGTT 2520
AGCAGAATGA ATCACCGATA CGCGAGCGAA CGTGAAGCGA CTGCTGCTGC AAAACGTCTG 2580
CGACCTGAGC AACAACATGA ATGGTCTTCG GTTTCCGTGT TTCGTAAAGT CTGGAAACGC 2640
GGAAGTCAGC GCCCTGCACC ATTATGTTCC GGATCTGCAT CGCAGGATGC TGCTGGCTAC 2700
CCTGTGGAAC ACCTACATCT GTATTAACGA AGCGCTGGCA TTGACCCTGA GTGATTTTTC 2760
TCTGGTCCCG CCGCATCCAT ACCGCCAGTT GTTTACCCTC ACAACGTTCC AGTAACCGGG 2820
CATGTTCATC ATCAGTAACC CGTATCGTGA GCATCCTCTC TCGTTTCATC GGTATCATTA 2880
CCCCCATGAA CAGAAATTCC CCCTTACACG GAGGCATCAA GTGACCAAAC AGGAAAAAAC 2940
CGCCCTTAAC ATGGCCCGCT TTATCAGAAG CCAGACATTA ACGCTTCTGG AGAAACTCAA 3000
CGAGCTGGAC GCGGATGAAC AGGCAGACAT CTGTGAATCG CTTCACGACC ACGCTGATGA 3060
GCTTTACCGC AGCTGCCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA 3120
GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA 3180
GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC ATGACCCAGT CACGTAGCGA 3240
TAGCGGAGTG TATACTGGCT TAACTATGCG GCATCAGAGC AGATTGTACT GAGAGTGCAC 3300
CATATGCGGT GTGAAATACC GCACAGATGC GTAAGGAGAA AATACCGCAT CAGGCGCTCT 3360
TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA 3420
GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC 3480
ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT 3540
TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG 3600
CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC 3660
TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC 3720
GTGGCGCTTT CTCAATGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC 3780
AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC 3840
TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT 3900
AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT 3960
AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC 4020
TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT 4080
TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG 4140
ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC 4200
ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA 4260
TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG 4320
GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG 4380
TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA 4440
GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG 4500
CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA 4560
GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTGCAGGC 4620
ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA 4680
AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG 4740
ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT 4800
AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC 4860
AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAACACGG 4920
GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG 4980
GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT 5040
GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA 5100
GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA 5160
CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC 5220
ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA 5280
GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT TAACCTATAA AAATAGGCGT 5340
ATCACGAGGC CCTTTCGTCT TCAAGAATTA ATTGTTATCC GCTCACAATT AATTCTTGAC 5400
AATTAGTTAA CTATTTGTTA TAATGTATTC ATAAGCTT 5438
【０１０７】
配列番号：13
配列の長さ：20
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
GCTGCCGAAC AGAAGTCTAA 20
【０１０８】
配列番号：14
配列の長さ：20
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
CTCGAAGGAA CAATGGATAC 20
【０１０９】
配列番号：15
配列の長さ：23
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
GTACATATTG TCGTTAGAAC GCG 23
【０１１０】
配列番号：16
配列の長さ：23
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
TAATACGACT CACTATAGGG AGA 23
【０１１１】
配列番号：17
配列の長さ：28
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
GCGGATCCTG ATGTCTATTT CATCTTCT 28
【０１１２】
配列番号：18
配列の長さ：30
配列の型：核酸
鎖の数：一本鎖
配列の種類：他の核酸 合成ＤＮＡ
配列
ATCTCGAGTT TTATGCTGCT GCGCCAGCGA 30[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring an anti-Chlamydia pneumoniae antibody, a reagent therefor, and a diagnostic agent for Chlamydia pneumoniae infection. 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 bacterium that causes trachoma, sexually transmitted lymphogranulomas, genitourinary infections, inclusion conjunctivitis, neonatal pneumonia, 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]
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.
On the other hand, pBBK10MM is known as a plasmid capable of expressing a large amount of protein in E. coli (Japanese Patent Laid-Open No. 4-117284). This plasmid can express a fusion protein of dihydrofolate reductase (hereinafter abbreviated as DHFR) and an antiallergic peptide. Since the fusion protein obtained here retains the enzyme activity of DHFR, purification of the fusion protein can be easily performed using the properties and activities of DHFR.
[0006]
[Problems to be solved by the invention]
The invention described in claim 1 provides a method for measuring an anti-Chlamydia pneumoniae antibody capable of specifically measuring an antibody against Chlamydia pneumoniae.
The invention described in claim 2 provides a highly sensitive measurement method for anti-Chlamydia pneumoniae antibodies in addition to the effects of the invention described in claim 1.
The invention described in claim 3 provides a method for measuring an anti-Chlamydia pneumoniae antibody having excellent antigen stability and high reliability in addition to the effects of the invention described in claim 1.
The invention described in claim 4 provides a method for measuring an anti-Chlamydia pneumoniae antibody capable of accurately diagnosing Chlamydia pneumoniae infection in addition to the effects of the invention described in claim 1.
The invention described in claim 5 provides a method for measuring an anti-Chlamydia pneumoniae antibody capable of accurate diagnosis of Chlamydia pneumoniae infection in addition to the effects of the invention described in claim 1.
[0007]
The invention according to claim 6 provides a reagent for measuring an anti-Chlamydia pneumoniae antibody capable of specifically measuring an antibody against Chlamydia pneumoniae.
The invention described in claim 7 provides a reagent for measuring anti-Chlamydia pneumoniae antibody having high sensitivity in addition to the effect of the invention described in claim 6.
The invention described in claim 8 provides a reagent for measuring an anti-Chlamydia pneumoniae antibody having excellent stability and high reliability in addition to the effect of the invention described in claim 6.
The invention described in claim 9 provides a reagent for measuring an anti-Chlamydia pneumoniae antibody capable of accurately diagnosing Chlamydia pneumoniae infection in addition to the effect of the invention described in claim 6.
The invention described in claim 10 provides a reagent for measuring anti-Chlamydia pneumoniae antibody capable of accurately diagnosing Chlamydia pneumoniae infection in addition to the effects of the invention described in claim 6.
[0008]
The invention according to claim 11 provides a diagnostic agent for Chlamydia pneumoniae infection that can specifically measure an antibody against Chlamydia pneumoniae.
The invention according to claim 12 provides a highly sensitive diagnostic agent for Chlamydia pneumoniae infection in addition to the effect of the invention according to claim 11.
The invention described in claim 13 provides a diagnostic agent for Chlamydia pneumoniae infection having excellent stability and high reliability in addition to the effect of the invention described in claim 11.
The invention described in claim 14 provides a diagnostic agent for Chlamydia pneumoniae infection that enables accurate diagnosis of Chlamydia pneumoniae infection in addition to the effects of the invention described in Claim 11.
The invention described in claim 15 provides a diagnostic agent for Chlamydia pneumoniae infection that enables accurate diagnosis of Chlamydia pneumoniae infection in addition to the effects of the invention described in Claim 11.
[0009]
[Means for Solving the Problems]
The present inventors first extracted genomic DNA from Chlamydia pneumoniae, partially digested with restriction enzymes, and inserted this into a phage vector λgt11 DNA to create a genomic DNA library, which was infected with E. coli Y1090r- strain. Screening of infected E. coli colonies expressing Chlamydia pneumoniae antigen polypeptide using Chlamydia pneumoniae-specific monoclonal antibody, extracting λ phage from positive infected E. coli, and repeating this procedure to purify λ phage The DNA was obtained.
Then, a recombinant plasmid vector pCPN533T was constructed from the DNA of this λ phage and the above-described plasmid pBBK10MM using gene recombination technology, thereby completing the present invention.
[0010]
That is, the present invention relates to the following (1) to (15).
(1) Polypeptide A containing a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 2 bound to the peptide represented by SEQ ID NO: 1 directly or via an intervening amino acid sequence A method for measuring an anti-Chlamydia pneumoniae antibody, characterized by using a dihydrofolate reductase-Chlamydia pneumoniae antigen fusion protein as an antigen.
(2) The method for measuring an anti-Chlamydia pneumoniae antibody according to (1) above, wherein the polypeptide A is a polypeptide lacking an amino acid from the peptide represented by SEQ ID NO: 2.
(3) Polypeptide A is a polypeptide in which an amino acid in the peptide represented by SEQ ID NO: 2 is substituted with another amino acid or an amino acid is inserted into the peptide represented by SEQ ID NO: 2. The method for measuring an anti-Chlamydia pneumoniae antibody according to (1) above.
(4) The method for measuring an anti-Chlamydia pneumoniae antibody according to (1) above, wherein the fusion protein is the peptide represented by SEQ ID NO: 3.
(5) The method for measuring an anti-Chlamydia pneumoniae antibody according to (1) above, wherein the fusion protein is the peptide represented by SEQ ID NO: 4.
[0011]
(6) Polypeptide A containing a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 2 bound to the peptide represented by SEQ ID NO: 1 directly or via an intervening amino acid sequence A reagent for measuring an anti-Chlamydia pneumoniae antibody comprising a dihydrofolate reductase-Chlamydia pneumoniae antigen fusion protein as an antigen.
(7) The reagent for measuring an anti-Chlamydia pneumoniae antibody according to (6) above, wherein the polypeptide A is a polypeptide lacking an amino acid from the peptide represented by SEQ ID NO: 2.
(8) Polypeptide A is a polypeptide in which an amino acid in the peptide represented by SEQ ID NO: 2 is substituted with another amino acid or an amino acid is inserted into the peptide represented by SEQ ID NO: 2. The reagent for measuring an anti-Chlamydia pneumoniae antibody according to (6) above.
(9) The reagent for measuring an anti-Chlamydia pneumoniae antibody according to (6) above, wherein the fusion protein is a peptide represented by SEQ ID NO: 3.
(10) The reagent for measuring an anti-Chlamydia pneumoniae antibody according to (6) above, wherein the fusion protein is a peptide represented by SEQ ID NO: 4.
[0012]
(11) Polypeptide A containing a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 2 bound to the peptide represented by SEQ ID NO: 1 directly or via an intervening amino acid sequence , A diagnostic agent for Chlamydia pneumoniae infection comprising dihydrofolate reductase-Chlamydia pneumoniae antigen fusion protein as an active ingredient.
(12) The diagnostic agent for Chlamydia pneumoniae infection according to (11) above, wherein the polypeptide A is a polypeptide lacking an amino acid from the peptide represented by SEQ ID NO: 2.
(13) Polypeptide A is a polypeptide in which the amino acid in the peptide represented by SEQ ID NO: 2 is substituted with another amino acid or an amino acid is inserted into the peptide represented by SEQ ID NO: 2. The diagnostic agent for Chlamydia pneumoniae infection as described in (11) above.
(14) The diagnostic agent for Chlamydia pneumoniae infection according to (11) above, wherein the fusion protein is a peptide represented by SEQ ID NO: 3.
(15) The diagnostic agent for Chlamydia pneumoniae infection according to (11) above, wherein the fusion protein is the peptide represented by SEQ ID NO: 4.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
Chlamydia pneumoniae antigen
In the present invention, the Chlamydia pneumoniae antigen is a polypeptide comprising a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 2 (hereinafter referred to as the peptide having the antigenicity) "Polypeptide A").
Since a longer amino acid sequence derived from the Chlamydia pneumoniae antigen polypeptide contained in the peptide chain of polypeptide A can be expected to produce a highly sensitive antigen-antibody reaction, polypeptide A includes a peptide represented by SEQ ID NO: 2 A polypeptide comprising a sequence consisting of 20 or more consecutive amino acids is preferred, a polypeptide comprising a sequence comprising 100 or more amino acids is more preferred, and a sequence comprising 250 or more amino acids is included. More preferably, it is a polypeptide.
[0014]
Moreover, as long as the antigenicity as a Chlamydia pneumoniae antigen is retained, the polypeptide A may be a peptide lacking amino acids (for example, 1 to 250) from the peptide represented by SEQ ID NO: 2. When the number of amino acids to be deleted is too large, the antigenicity of polypeptide A as Chlamydia pneumoniae antigen tends to be impaired.
When the number of amino acids to be deleted is large (for example, 5 or more), the antigenicity as a Chlamydia pneumoniae antigen is likely to decrease. In order to minimize this decrease, the amino acid that is deleted from the peptide represented by SEQ ID NO: 2 Is preferably continuous (for example, 5 or more).
[0015]
Moreover, as long as the antigenicity as a Chlamydia pneumoniae antigen is retained, a polypeptide containing an amino acid sequence other than that derived from the Chlamydia pneumoniae antigen polypeptide can be used as the polypeptide A. However, this polypeptide is required to have no or low antigenicity as a substance other than Chlamydia pneumoniae antigen as compared to antigenicity as Chlamydia pneumoniae antigen.
Examples of such a polypeptide include a polypeptide in which an amino acid (for example, 1 to 100) in the peptide represented by SEQ ID NO: 2 is substituted with another amino acid, and a peptide represented by SEQ ID NO: 2 Examples include polypeptides into which amino acids (for example, 1 to 100) have been inserted.
[0016]
When the number of amino acids to be substituted or inserted is large (for example, 5 or more), the antigenicity as a Chlamydia pneumoniae antigen is likely to decrease. To minimize this decrease, the peptide of SEQ ID NO: 2 It is preferable that the amino acid to be substituted or inserted therein is continuous (for example, 5 or more).
The amino acid to be substituted is preferably one having similar properties, for example, substitution of glycine and alanine.
Specific examples of the polypeptide A include, for example, a peptide represented by SEQ ID NO: 2, a peptide in which the 488th amino acid of the peptide represented by SEQ ID NO: 2 is substituted with glycine, and a peptide represented by SEQ ID NO: 4. And a peptide consisting of the amino acid sequence of positions 162 to 432.
[0017]
Dihydrofolate reductase-Chlamydia pneumoniae antigen fusion protein used as antigen or active ingredient
The dihydrofolate reductase-Chlamydia pneumoniae antigen fusion protein used as an antigen or active ingredient is a peptide sequence represented by SEQ ID NO: 1, directly or via an intervening amino acid sequence. Polypeptide A containing a sequence consisting of at least 5 amino acids is bound.
The peptide represented by SEQ ID NO: 1 is a dihydrofolate reductase (DHFR) portion of the fusion protein.
Polypeptide A comprising a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 2 is the aforementioned Chlamydia pneumoniae antigen portion.
The intervening amino acid sequence is not particularly limited, and examples thereof include leucine and leucine-methionine amino acid sequences.
[0018]
Specific examples of the DHFR-Chlamydia pneumoniae antigen fusion protein of the present invention include, for example, peptides represented by SEQ ID NO: 3 and SEQ ID NO: 4.
The peptide represented by SEQ ID NO: 3 is a fusion protein in which the entire 53 KDa antigen polypeptide of Chlamydia pneumoniae is bound to DHFR, and possesses antigenicity as a Chlamydia pneumoniae antigen.
The peptide represented by SEQ ID NO: 4 is a fusion protein in which a part of the 53 kDa antigen polypeptide of Chlamydia pneumoniae and another peptide are bound to DHFR, and possesses antigenicity as a Chlamydia pneumoniae antigen.
Among the above fusion proteins, the peptide represented by SEQ ID NO: 3, which contains the entire Chlamydia pneumoniae 53 KDa antigen polypeptide, is preferred.
[0019]
Method for producing fusion protein used as antigen or active ingredient
Examples of the method for producing a DHFR-Chlamydia pneumoniae antigen fusion protein used as an antigen or active ingredient include a gene recombination method.
As a gene recombination method, for example, a DNA encoding the fusion protein is inserted into a vector to construct a recombinant vector, which is inserted into a host to produce a transformant. A method for purifying the fusion protein can be mentioned.
The DNA encoding the fusion protein 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 fusion protein using the transformant will be described in detail.
[0020]
Production of recombinant vector containing DNA encoding fusion protein and production of transformant containing the same
First, DNA encoding Chlamydia pneumoniae antigen polypeptide (described later) and DNA encoding DHFR (described later) are linked using a commercially available kit or the like. As a commercially available kit, for example, a DNA ligation kit (trade name, Takara Shuzo Co., Ltd.) can be used. If the DNA obtained by ligation does not have a replication origin and does not function as a plasmid or phage, this DNA is inserted into a new plasmid vector or phage vector to construct a recombinant vector. As a new plasmid vector, for example, pBR322, pUC18, pUC19 and the like can be used. In any case, the insertion site of DNA encoding the fusion protein is required to be downstream of the promoter region.
[0021]
The ligation reaction product or the obtained recombinant vector is put into a host to produce a transformant. When using an E. coli-derived plasmid or λ phage, 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 Co., Ltd. A general method for preparing a transformant by putting the ligation reaction product into a host is “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".
[0022]
The obtained 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 a desired recombinant plasmid. Select. Examples of the plasmid vector thus prepared include pCPN533T plasmid.
Examples of the transformant thus prepared include Escherichia coli HB101 strain containing the aforementioned recombinant vector pCPN533T. Escherichia coli containing pCPN533T has been deposited at the Institute of Biotechnology, Industrial Technology Institute, under the accession number FERM BP-5222.
[0023]
As a method for culturing the transformant, for example, a method in which the incubator is shaken at an appropriate temperature until the fusion protein of interest is sufficiently accumulated in the transformant in a medium in which the transformant can grow is used. Can do. When using E. coli HB101 strain containing the above-mentioned recombinant vector pCPN533T as a transformant, the culture is shaken overnight at 37 ° C. in LB medium containing ampicillin, and then this culture solution is TB containing ampicillin and trimethoprim. A method of inoculating a medium or the like and further culturing overnight at 37 ° C. with shaking can be used. The preparation method of TB medium is described in the document “Molecular Cloning”.
[0024]
As a method for disrupting the cultured transformant, for example, the transformant is collected by centrifugation, suspended in a buffer solution to produce a suspension, and a physical impact is applied to the suspension. Can be adopted. As the buffer solution, for example, a TE buffer solution can be used. As a method of giving a physical impact to the suspension, for example, a method of irradiating the suspension with ultrasonic waves can be used.
When the transformant is E. coli, instead of giving a physical impact to the suspension, for example, lysozyme is added to the suspension, and TE buffer containing sodium dodecyl sulfate (SDS) is added and stirred. Thus, a method of dissolving the transformant can also be employed.
The transformant is crushed or dissolved, and then centrifuged to remove cell debris and obtain a supernatant.
[0025]
As a method for purifying the fusion protein, for example, each step of removing a nucleic acid to which streptomycin sulfate is added and obtaining a protein to which ammonium sulfate is added can be used.
As the step of removing the nucleic acid by adding streptomycin sulfate, for example, adding streptomycin sulfate to any of the supernatants described above, stirring for a while and centrifuging to remove the nucleic acid as a precipitate, An operation to obtain the supernatant can be used.
As the step of obtaining protein by adding ammonium sulfate, for example, an operation of adding ammonium sulfate to the supernatant after removing nucleic acid as a precipitate, stirring, and centrifuging can be used. Usually, the precipitate is obtained, but the target fusion protein may be contained in the supernatant, and it is preferable to sample to confirm the presence or absence of the target fusion protein.
[0026]
Then, the process of acquiring the fraction containing the target fusion protein is performed. In this step, for example, the precipitate is dissolved in a small amount of buffer or the supernatant is fractionated by liquid chromatography, and the fraction containing the target fusion protein is identified and the fraction is separated. A method of getting minutes can be used. Examples of a method for identifying the fraction containing the target fusion protein include Western blotting using a Chlamydia pneumoniae-specific monoclonal antibody (described later). Specific methods for removing cell debris, removing DNA to which streptomycin sulfate is added, obtaining a protein to which ammonium sulfate is added, and Western blotting are described in the document "Molecular Cloning".
[0027]
On the other hand, a method for obtaining a fraction containing the target fusion protein using the affinity of DHFR can also be used. In this method, for example, the step of flowing the supernatant or the like on a methotrexate-agarose column, adsorbing the fusion protein to the column, and then eluting the fusion protein using an eluate containing folic acid can be used.
[0028]
DNA encoding the fusion protein
In the present invention, the DNA encoding the fusion protein refers to the amino acid sequence of the fusion protein of the present invention in accordance with the triplet code table (1 to 6 nucleotide sequences are assigned to each amino acid). It is a DNA selected from a DNA group when read into a nucleotide sequence.
Among fusion proteins, specific examples of the base sequence of the DHFR part include the 1st to 480th base sequences of the base sequence represented by SEQ ID NO: 5. On the other hand, specific examples of the base sequence of the Chlamydia pneumoniae antigen part include the 484th to 1947th base sequences of the base sequence represented by SEQ ID NO: 5. The 481-483rd base sequence of the base sequence shown in SEQ ID NO: 5 is the base sequence of the intervening amino acid sequence.
[0029]
Specific examples of DNA encoding the fusion protein include the base sequence represented by SEQ ID NO: 5 and the base sequence represented by SEQ ID NO: 6.
The base sequence shown in SEQ ID NO: 5 is DNA encoding a fusion protein of the entire DHFR and Chlamydia pneumoniae 53KDa antigen polypeptide, and the base sequence shown in SEQ ID NO: 6 is DHFR and Chlamydia pneumoniae 53KDa antigen poly It is DNA encoding a peptide (partial) fusion protein.
[0030]
DNA encoding the fusion protein can be prepared by a genetic recombination method.
Examples of the gene recombination method include, as described above, a method in which a DNA encoding a Chlamydia pneumoniae antigen polypeptide and a DNA encoding DHFR are separately obtained and both are linked by a gene recombination method. .
DNA encoding DHFR can be excised from a plasmid vector containing the DNA using restriction enzymes, or a plasmid DNA or genomic DNA containing the DNA can be used as a template, and PCR can be performed using appropriate primers. It can be obtained by amplifying the DNA.
[0031]
In the former method, for example, plasmid vector pBBK10MM or pCPN533T which is also the recombinant vector of the present invention can be used as a plasmid vector containing DNA encoding DHFR. In order to obtain a plasmid from Escherichia coli (plasmid-carrying bacteria) containing pCPN533T, a normal method for obtaining plasmid DNA may be followed, and this method is described in the document “Molecular Cloning”. When plasmid pBBK10MM is used, BamHI and XhoI are used as restriction enzymes, and a DNA fragment of about 4.6 Kbp may be excised.
In the latter method, for example, the above-described pBBK10MM and pCPN533T can be used as they are as plasmid DNA, and, for example, Bacillus subtilis genomic DNA can be used. In order to obtain genomic DNA, a normal method for obtaining genomic DNA may be followed, and this method is described in the document "Molecular Cloning".
[0032]
Primers used in the latter method can be designed and synthesized in consideration of the nucleotide sequences at the 5 ′ end and 3 ′ end of DNA encoding DHFR. For example, an oligonucleotide having the 1st to 20th sequences of the base sequence of SEQ ID NO: 5 and an oligonucleotide having a sequence complementary to the 461st to 480th sequences can be used. These oligonucleotides can be chemically synthesized using a commercially available DNA synthesizer.
[0033]
Next, a method for cloning DNA encoding Chlamydia pneumoniae antigen polypeptide from Chlamydia pneumoniae will be described in detail.
Chlamydia pneumoniae culture
Cultured HL cells and the like are infected with Chlamydia pneumoniae in advance, and the cells are treated with SPG solution (sucrose 75.0 g, phosphate 1 potassium 0.52 g, phosphate dipotassium 1.22 g and glutamate 0.72 g in water. Suspend in an aqueous solution dissolved in 1 liter, pH 7.4 to 7.6), crush or dissolve this animal cell, and centrifuge to obtain a supernatant (Chlamydia pneumoniae suspension). 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)).
A cell suspension is prepared from cultured HL cells, and after removing the culture supernatant, the above-mentioned Chlamydia pneumoniae suspension is added, cultured, centrifuged, and Chlamydia pneumoniae propagated in the cell. Obtain HL cells.
[0034]
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. Since it has been confirmed with an electron microscope that the basic body of Chlamydia pneumoniae is contained in a yellowish white band in a preliminary experiment, this band is collected.
[0035]
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, and Chlamydia pneumoniae genomic DNA is obtained.
[0036]
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.
[0037]
This DNA fragment was reacted with λgt11 DNA, ATP and T4 ligase that 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.
[0038]
Cloning of DNA encoding Chlamydia pneumoniae antigen polypeptide The above-mentioned genomic DNA expression library is infected with the culture solution of Escherichia coli Y1090r- strain, cultured on an agar medium, and added to an aqueous isopropylthio-β-D-galactoside (IPTG) solution. Using the soaked nitrocellulose filter, the protein produced in the cells by the expression of the inserted DNA 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). Has been described. 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.
[0039]
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 polypeptide used as the antigen or active ingredient is cloned to express the polypeptide reactive with Chlamydia pneumoniae-specific monoclonal antibody. Obtain λ phage.
[0040]
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 are trade names manufactured by Takara Shuzo Co., Ltd.). Examples of the tack polymerase include an amplifier. There is Retac DNA Polymerase (trade name, manufactured by Takara Shuzo Co., Ltd.). A general method of this DNA amplification method is known as a PCR method, and details are described in the document "Molecular Cloning".
[0041]
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 (trade name, manufactured by 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.
[0042]
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.
Once DNA encoding a polypeptide used as an antigen or active ingredient is obtained, the DNA can be replicated by using the genetic recombination method or the PCR method described above, so that Chlamydia pneumoniae There is no need to reacquire DNA encoding the Chlamydia pneumoniae antigen polypeptide from the basic body.
[0043]
The method using a gene recombination method can be performed as follows, for example. First, as described above, the obtained DNA is inserted into an existing plasmid vector or phage vector to prepare a recombinant vector, and the recombinant vector is put into a host to produce a transformant. Incubate the body. Since the recombinant vector is amplified in the transformant by this culture, the amplified recombinant vector is obtained from the transformant, and the DNA is cut out using a restriction enzyme. The operation for obtaining the recombinant vector amplified from the transformant can be performed, for example, as follows. When the recombinant vector is a plasmid, the transformant is crushed and subjected to phenol / chloroform treatment and ethanol precipitation treatment to obtain DNA. The obtained DNA is ultracentrifuged using ethidium bromide-containing cesium chloride, and the recombinant vector is obtained as plasmid DNA. On the other hand, when the recombinant vector is a phage, the transformant is disrupted, phage particles are obtained, the phage particles are dissolved, and the recombinant vector is obtained as phage DNA.
[0044]
As a method using the PCR method, for example, a primer DNA is prepared by a chemical synthesis method based on the base sequences at both ends of the DNA to be amplified, and a DNA encoding a Chlamydia pneumoniae antigen polypeptide is used as a template DNA. A method for performing PCR can be used.
General techniques for replicating DNA using genetic recombination methods or PCR methods are described in the document "Molecular Cloning".
[0045]
Measuring method and reagent for anti-Chlamydia pneumoniae antibody and diagnostic agent for Chlamydia pneumoniae infection
The anti-Chlamydia pneumoniae antibody can be measured, for example, by immobilizing the antigen or the fusion protein used as an active ingredient on a carrier, adding a specimen, washing, adding a labeled secondary antibody, and washing. A method of directly or indirectly measuring this label can be used.
Examples of the carrier include particles, threads, plates (assay plates, etc.), and examples of these materials include latex, cellulose, plastics (polystyrene, etc.), and the like.
In order to immobilize the fusion protein on a carrier, for example, covalent bond or physical adsorption can be used.
[0046]
As the specimen, for example, human serum or the like can be used. In order to prevent other antibodies in the sample from binding nonspecifically to the carrier, it is preferable to block the surface of the carrier with bovine serum albumin or the like before the sample is added.
The washing can be performed using, for example, a phosphate buffer containing a surfactant.
[0047]
Examples of the labeled secondary antibody include a labeled anti-human monoclonal antibody. Various labels can be used, such as enzymes (alkaline phosphatase, luciferase, peroxidase, β-galactosidase, etc.), fluorescent substances (fluorescine, etc.) ) Can be used. In addition, a chemical substance (biotin, avidin, streptavidin, digoxigenin, etc.) may be interposed between the antibody and the label.
As a method for directly or indirectly measuring the label, for example, when the label is an enzyme, a substrate is added, and light or color generated by the catalytic action of the enzyme is measured, or a change in absorbance is measured. A method is available. When the label is a fluorescent substance, a method of irradiating the reaction system with ultraviolet rays and measuring the generated fluorescence can be used. It is preferable to use a sensitizer if necessary.
In the present invention, “measurement” means not only quantitative or semi-quantitative measurement but also qualitative measurement (detection, etc.).
[0048]
Examples of the reagent for measuring anti-Chlamydia pneumoniae antibody using the fusion protein as an antigen include, for example, those obtained by immobilizing the fusion protein on a carrier, and encapsulating the necessary amount of the labeled secondary antibody, substrate, etc. The thing which was done is mentioned. As a diagnostic agent for Chlamydia pneumoniae infection using the above fusion protein as an active ingredient, for example, the above reagent can be used as it is.
Examples of the reagent for measuring anti-Chlamydia pneumoniae antibody comprising the fusion protein as an antigen include, for example, a carrier on which the fusion protein is immobilized, the labeled secondary antibody, a substrate and the like in a necessary amount. Kits, bulks of these single items, and the like can be mentioned.
As a diagnostic agent for Chlamydia pneumoniae infection using the above fusion protein as an active ingredient, for example, the above reagent can be used as it is.
[0049]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited at all by this.
In the following examples, the monoclonal antibodies used are SCP53 and AY6E2E8. SCP53 is an antibody secreted by hybridoma SCP53 obtained by one of the present inventors Matsumoto et al. Immunizing mice using Chlamydia pneumoniae KKpn-1 strain as an antigen and fusing the spleen cells with myeloma cells, In addition, AY6E2E8 is a hybridoma AY6E2E8 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. Is the antibody AY6E2E8 secreted by
As shown in Table 1, the monoclonal antibodies SCP53 and AY6E2E8 are specific for C. pneumoniae.
[0050]
[Table 1]

[0051]
A method for producing a monoclonal antibody will be described later.
The following description will be made in order from the culture of Chlamydia pneumoniae host cells to the determination of the gene DNA sequence and amino acid sequence of the Chlamydia pneumoniae antigen polypeptide.
[0052]
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. 4 ml of a mixture of 8 ml of this cell suspension and 292 ml of Dulbecco's MEM medium containing 10% fetal bovine serum was added to each well of a 6-well plastic culture vessel and cultured in a 5% (v / v) carbon dioxide atmosphere.
[0053]
(B) Culture of Chlamydia pneumoniae YK-41
As Chlamydia, Chlamydia pneumoniae YK-41 strain (Kanemoto et al .: Microbiol. Immunol., Vol. 37, P.495-498, 1993) was used.
The obtained HL cells are pre-infected with Chlamydia pneumoniae YK-41 strain, suspended in SPG solution, placed in a polypropylene centrifuge tube, and irradiated with ultrasonic waves 30 times at 1 second intervals. The tube was centrifuged at 1,500 × g for 3 minutes, and the supernatant was obtained to obtain a Chlamydia pneumoniae suspension.
The culture supernatant of HL cells grown in a 6-well plastic culture vessel (on the bottom) was removed with a pipette, 2 ml of Chlamydia pneumoniae suspension was added to each well, and centrifugal adsorption was performed 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.
[0054]
(C) Purification of the basic body of Chlamydia pneumoniae YK-41
The frozen HL cell suspension of Chlamydia pneumoniae YK-41 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.
[0055]
Separately, 0.03M Tris-HCl buffer (pH 7.4) containing 50% (w / v) sucrose in a centrifuge tube, then 3 volumes of urografin 76% (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 are collected, and the same volume of SPG solution is added to the collected solution, and centrifuged at 10,000 rpm for 30 minutes. did. The supernatant was discarded and the precipitate was suspended in the SPG solution. Prepare a continuous density gradient liquid from 35% to 50% (volume ratio of the former with respect to the total volume) of urografin 76% (manufactured by Schering) and 0.03M Tris-HCl buffer (pH 7.4) in a centrifuge tube. 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 collected, diluted twice with SPG solution, and centrifuged at 10,000 rpm for 30 minutes. The obtained precipitate was suspended in an SPG solution, and the protein concentration was measured (using a protein measurement kit manufactured by Bio-Rad, with bovine serum albumin as a standard), and then stored at -70 ° C.
[0056]
(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 0.1 M 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.
[0057]
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, and then centrifuged at 12,000 rpm for 5 minutes at 4 ° C. 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.
[0058]
(E) Preparation of genomic DNA expression library
To 100 μl of genomic DNA solution, 10 μl of M-buffer for restriction enzymes and 10 μl of restriction enzyme mixture (AccI, HaeIII and 0.4 μl each of AluI diluted 1/50 and 20 μl TE) 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.
[0059]
The obtained partially digested DNA is dissolved in 20 μl of purified water, and 19 μl thereof is taken, and 14 μl of a linker (20 pmole / μl) shown in the following chemical formula 1 (10 μm), 4.5 μl of 10 mM ATP, 50 mM MgCl ₂ Add 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 The mixture was reacted at 16 ° C. for 4 hours to add a linker.
[Chemical 1]

[0060]
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 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 .
[0061]
To 0.6 μl of this solution, 1 μl of λgt11 DNA (1 μg / μl, Stratagene) previously cleaved with restriction enzyme EcoRI, 0.5 μl of 10-fold concentration ligation buffer, 0.5 μl of 10 mM ATP, T4 ligase 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).
[0062]
(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 bovine 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.
[0063]
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. Furthermore, 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.
[0064]
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.
[0065]
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 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 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.
[0066]
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.
[0067]
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 hybridoma AY6E2E8.
[0068]
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 a plastic cell culture flask 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, Balb / c mice that had been injected intraperitoneally with 0.5 ml of pristane 2 weeks ago were injected with 1 × 10 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.
[0069]
Purification of monoclonal antibodies
The monoclonal antibody produced by the hybridoma AY6E2E8 was purified as follows. 3 volumes of PBS was added to 1 volume of monoclonal antibody-containing ascites obtained by intraperitoneal injection of hybridoma AY6E2E8, and the mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was 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 allowed to flow at 1 ml / min for 3 minutes, then 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.
[0070]
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 was obtained in the same manner as hybridoma AY6E2E8. As a result of examining the specificity of the monoclonal antibody produced by the hybridoma SCP53 in the same manner as described above, it was found that this monoclonal antibody recognizes the 53 KDa antigen polypeptide of Chlamydia pneumoniae.
Further, as a result of examining the subclasses of the monoclonal antibodies produced by the hybridoma AY6E2E8 and the hybridoma SCP53, the subclasses of these antibodies are IgG, respectively. ₂ b and IgG ₁ Met.
[0071]
(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 of 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 overnight at 37 ° C. Centrifugation was performed at 2,000 rpm for 10 minutes. 10 mM MgSO for precipitation (E. coli) _Four Add 9 ml of aqueous solution, mix, take 0.35 ml of this E. coli suspension, add 0.1 μl of λgt11 (DNA library) suspension, incubate at 37 ° C. for 20 minutes, and add λgt11 to E. coli. Infected. 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 a 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.
[0072]
The filter is immersed in a 0.1% bovine serum albumin-containing solution of 150 mM NaCl-containing 20 mM Tris-HCl buffer (pH 7.5) (hereinafter referred to as TBS buffer) 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 μg / ml of Chlamydia pneumoniae-specific monoclonal antibody (AY6E2E8 or SCP53), 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.
[0073]
Plaques on the agar medium corresponding to the colored spots on the filter were searched and identified, and the agar in this portion 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.
[0074]
(H) Culture of 53-3Sλ phage and DNA purification
Plaques were formed in the same manner as described in (F) above, and one plaque was collected, placed in 100 μl of SM buffer, and left overnight at 4 ° C. to extract λ phage. 5 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 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 the Wizard λ preps kit (Promega, trade name).
[0075]
(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 (trade name, manufactured by Takara Shuzo Co., Ltd.) 1 μl, 20 nM λgt11 reverse primer (Takara Shuzo Co., Ltd., trade name) 1 μl and AmpliTaq DNA Polymerase (Takara Shuzo Co., Ltd., trade name) 0.5 μl were added, and two 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 a Wizard PCR Prep kit (trade name, manufactured by Promega).
[0076]
(J) DNA sequence analysis
DNA base sequence analysis uses a DNA amplified by PCR as a template, performs a sequence reaction by a fluorescence-labeled terminator cycle sequence method using Taq DNA polymerase, and analyzes with a 373A type DNA sequencer (product name, Applied Biosystems). went. Using the DNA sequence analysis software “DNASIS” (trade name, manufactured by Hitachi Software Engineering Co., Ltd.), the obtained DNA base sequence was edited, linked, and the amino acid translation region was estimated to obtain the sequence of SEQ ID NO: 7. .
The analysis result of the sequence of SEQ ID NO: 7 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.
[0077]
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: 7 with the GenBank database, there was no known sequence showing high homology.
[0078]
Example 2 Production of Recombinant Vector Containing DNA Encoding Fusion Protein Containing DHFR and Chlamydia pneumoniae 53KDa Antigen Polypeptide and Transformant Containing It
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.
On the other hand, 53-3Sλ phage DNA was cleaved with the restriction enzyme EcoRI, and a DNA fragment of about 1.0 Kbp was similarly purified, and then further cleaved with the restriction enzyme AvaII, and a DNA fragment of about 0.8 Kbp was similarly purified. Add 100 ng of the above-mentioned DNA fragment of about 0.8 Kbp and 1 ng of each of the synthetic DNAs of SEQ ID NOs: 8 to 11 to 100 ng of the above-mentioned DNA fragment of about 4.6 Kbp, and link these DNAs using a DNA ligation kit (Takara Shuzo). did. This reaction product was put into Escherichia coli HB101 strain competent cell (Takara Shuzo) to prepare a transformant.
[0079]
This transformant was applied to an LB agar medium containing 50 mg / L ampicillin and cultured at 37 ° C. for 24 hours. The obtained colonies of E. coli were inoculated into 3 ml of LB medium containing 50 mg / L ampicillin and cultured overnight at 37 ° C. with shaking. The plasmid vector was isolated by the alkaline lysis method, cleaved with the restriction enzyme NruI, analyzed by 0.8% agarose gel electrophoresis, and Escherichia coli having a recombinant plasmid vector in which 616 bp and 4822 bp DNA fragments were generated was selected. The resulting recombinant plasmid vector was named pCPN533T. This plasmid vector is an approximately 5.4 kbp DNA having the base sequence of SEQ ID NO: 12, and expresses a fusion protein in which a polypeptide containing a part of the 53 KDa antigen polypeptide of Chlamydia pneumoniae is linked to the C-terminus of DHFR. Is. The base sequence of the DNA encoding the fusion protein is as shown in SEQ ID NO: 6, and the amino acid sequence deduced from this base sequence is as shown in SEQ ID NO: 4.
[0080]
Example 3 Identification of a fusion protein of a polypeptide containing a portion of the 53 KDa antigen polypeptide of DHFR and Chlamydia pneumoniae
One platinum loop of E. coli HB101 strain carrying plasmid pCPN533T was inoculated into 3 ml of LB medium containing 50 mg / l ampicillin and cultured with shaking at 37 ° C. overnight. 10 μl of loading buffer (0.0156 bromophenol blue, 10% mercaptoethanol, 20% glycerol, 0.156 M Tris-HCl buffer, pH 6.8 containing 5% SDS) is added to 10 μl of the medium containing E. coli, After heating at 80 ° C. for 5 minutes, the reaction solution was subjected to 5-20% polyacrylamide gradient gel electrophoresis. One sheet of filter paper moistened with 0.3 M Tris aqueous solution containing 10% methanol and 0.05% sodium dodecyl sulfate on the anode plate of the semi-driving device 25 mM Tris containing 10% methanol and 0.05% sodium dodecyl sulfate One filter paper moistened with aqueous solution, one nitrocellulose membrane moistened with 25 mM Tris aqueous solution containing 10% methanol, 0.05% sodium dodecyl sulfate, 40 mM aminocaproic acid, polyacrylamide gel after completion of the electrophoresis, 40 mM Two filter papers moistened with 25 mM Tris aqueous solution containing aminocaproic acid are stacked in this order, and the cathode plate is set to 2.5 mA / cm. ² The protein in the polyacrylamide gel was transferred to a nitrocellulose membrane by applying a current for 1 hour at a current density of. This nitrocellulose membrane was placed in a TBS buffer containing 0.1% bovine serum albumin and left standing at room temperature for 1 hour or longer to block. The nitrocellulose membrane was washed twice with TTBS buffer, and then shaken at 37 ° C. for 1 hour in a monoclonal antibody solution (in 10 μg / ml TTBS buffer) produced by hybridoma AY6E2E8. The nitrocellulose membrane was washed 3 times with TTBS buffer, and then shaken in a peroxidase-labeled anti-mouse IgG antibody solution (in 50 ng / ml TTBS buffer) at 37 ° C. for 1 hour. The nitrocellulose membrane is washed 3 times with TTBS buffer, and then added to a chromogenic substrate solution (100 ml of TBS buffer is mixed with 60 μl of 30% aqueous hydrogen peroxide and 20 ml of 4-chloro-1-naphthol methanol solution). And reacted at room temperature for 30 minutes. The nitrocellulose membrane was taken out, washed with purified water and air-dried. As a result, a colored band was observed at a position corresponding to the size of the fusion protein, and Escherichia coli having the plasmid pCPN533T expressed a 53 KDa antigen capable of reacting with a monoclonal antibody that reacts specifically with Chlamydia pneumoniae. It was shown that.
[0081]
Example 4 Acquisition of DNA encoding the entire Chlamydia pneumoniae 53 KDa antigen polypeptide
Based on the nucleotide sequence of SEQ ID NO: 7, DNAs having the nucleotide sequences of SEQ ID NOS: 13 and 14 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) and 15 μl of ligation solution in PCR in vitro Cloning Kit (trade name, manufactured by Takara Shuzo Co., Ltd.) were added and incubated at 16 ° C. for 30 minutes.
[0082]
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 base sequence of SEQ ID NO: 15 (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.
[0083]
In 1 μl of the reaction solution after the PCR step, 1 μl of DNA having the base sequence of SEQ ID NO: 14 (20 pmol / μl) and DNA having the base sequence of SEQ ID NO: 16 (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, trade name) 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.
[0084]
Using the obtained aqueous DNA solution, a sequencing reaction is performed by a fluorescence-labeled terminator cycle sequencing method using Taq DNA polymerase using the contained DNA as a template, and the DNA of the DNA is obtained using a 373A type DNA sequencer (product name, Applied Biosystems). The nucleotide sequence was analyzed. Using the DNA sequence analysis software “DNASIS” (manufactured by Hitachi Software Engineering Co., Ltd., trade name), 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.
[0085]
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 nucleotide sequence of SEQ ID NO: 7, as a primer corresponding to the upstream portion of the DNA encoding the Chlamydia pneumoniae antigen polypeptide, the DNA having the nucleotide sequence of SEQ ID NO: 17 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: 18 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.
[0086]
Using 1 μl of an aqueous solution of Chlamydia pneumoniae YK41 genomic DNA obtained in Example 1 as a primer DNA, 1 μl of DNA having the base sequence of SEQ ID NO: 17 (20 pmol / μl) and DNA having the base sequence of SEQ ID NO: 18 (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 nucleotide sequence of the obtained DNA, it was found that this DNA has the nucleotide sequence of 484 to 1947 of SEQ ID NO: 5 and encodes the amino acid sequence of 162 to 649 of SEQ ID NO: 3. It was.
Further, genome walking was performed using the plasmid pCPN533T and the above-described λgt11 DNA library to obtain DNA encoding the entire 53 KDa antigen polypeptide of Chlamydia pneumoniae.
[0087]
Example 5 Production of a recombinant vector containing DNA encoding a fusion protein of the entire 53 KDa antigen polypeptide of DHFR and Chlamydia pneumoniae and production of a transformant containing the same
Recombinant vector comprising DNA encoding fusion protein of DHFR and Chlamydia pneumoniae 53KDa antigen polypeptide in the same manner as in Example 2 using plasmid pBBK10MM and DNA encoding the entire Chlamydia pneumoniae 53KDa antigen polypeptide And a transformant containing the same. The base sequence of DNA encoding this fusion protein is as shown in SEQ ID NO: 5, and the amino acid sequence deduced from this base sequence is as shown in SEQ ID NO: 3.
[0088]
Example 6 Measurement of anti-Chlamydia pneumoniae antibody using fusion protein as antigen
Measurement of anti-Chlamydia pneumoniae antibody using the fusion protein as an antigen can be performed as follows.
As a fusion protein, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 was used, dissolved in PBS, poured into a well of a microtiter plate, and allowed to stand overnight at 4 ° C. Fix the titer plate (physical adsorption). Subsequently, PBS containing bovine serum albumin is added, and the mixture is allowed to stand overnight at 4 ° C. for blocking. After removing PBS containing bovine serum albumin and washing twice with PBS containing 0.02% (w / v) Tween 20, the patient's serum is added to the wells and left at room temperature for 2 hours. After washing with PBS containing 0.02% (w / v) Tween 20 three times in the same manner, a peroxidase-labeled mouse anti-human IgG antibody is added to the wells and left at room temperature for 2 hours. After washing three times with PBS containing 0.02% (w / v) Tween 20, add ABTS solution (manufactured by KPL) to the wells, and let stand for 15 minutes to 1 hour at room temperature for color reaction, 96 wells Absorbance at 405 nm is measured with a photometer for EIA plates.
[0089]
【The invention's effect】
The method for measuring an anti-Chlamydia pneumoniae antibody according to claim 1 can specifically measure an antibody against Chlamydia pneumoniae, and is suitable for an antibody test for Chlamydia pneumoniae and a diagnosis of Chlamydia pneumoniae infection.
The method for measuring an anti-Chlamydia pneumoniae antibody according to claim 2 has the effect of the method according to claim 1, and further, since the amino acid sequence of the fusion protein used as the antigen is short, it is immobilized on a carrier or the like The number of antigen peptides that can be increased can be increased, and the sensitivity is high.
The method for measuring an anti-Chlamydia pneumoniae antibody according to claim 3 has the effect of the measurement method according to claim 1, and can further form a structure in which a fusion protein used as an antigen is not easily degraded by a proteolytic enzyme. Therefore, it is excellent in stability and the reliability of the measurement result is high.
[0090]
The method for measuring an anti-Chlamydia pneumoniae antibody according to claim 4 has the effect of the method according to claim 1, and further, since the fusion protein used as an antigen has the entire antigen polypeptide specific for Chlamydia pneumoniae. It is extremely suitable for antibody testing and accurate diagnosis of Chlamydia pneumoniae infection.
The method for measuring an anti-Chlamydia pneumoniae antibody according to claim 5 has the effect of the measurement method according to claim 1, and further, since the fusion protein used as an antigen has an antigen part specific to Chlamydia pneumoniae, It is extremely suitable for testing and accurate diagnosis of Chlamydia pneumoniae infection.
[0091]
The reagent for measuring an anti-Chlamydia pneumoniae antibody according to claim 6 can specifically measure an antibody against Chlamydia pneumoniae, and is suitable for an antibody test for Chlamydia pneumoniae and a diagnosis of Chlamydia pneumoniae infection.
The reagent for measuring anti-Chlamydia pneumoniae antibody according to claim 7 has the effect of the reagent for measurement according to claim 6, and further, since the amino acid sequence of the fusion protein used as the antigen is short, The number of antigen peptides that can be immobilized can be increased, and the sensitivity is high.
The reagent for measuring anti-Chlamydia pneumoniae antibody according to claim 8 exhibits the effect of the reagent for measuring according to claim 6, and further, the fusion protein used as an antigen has a structure that is difficult to be degraded by proteolytic enzymes. Therefore, it has excellent stability and high reliability of measurement results.
[0092]
The reagent for measuring anti-Chlamydia pneumoniae antibody according to claim 9 exhibits the effect of the reagent for measurement according to claim 6, and further, the fusion protein used as the antigen is an entire antigen polypeptide specific for Chlamydia pneumoniae. Therefore, it is extremely suitable for antibody testing and accurate diagnosis of Chlamydia pneumoniae infection.
The measurement reagent for anti-Chlamydia pneumoniae antibody according to claim 10 has the effect of the measurement reagent according to claim 6, and further, since the fusion protein used as an antigen has an antigen part specific to Chlamydia pneumoniae. It is extremely suitable for antibody testing and accurate diagnosis of Chlamydia pneumoniae infection.
[0093]
The diagnostic agent for Chlamydia pneumoniae infection according to claim 11 can specifically measure an antibody against Chlamydia pneumoniae and is suitable for diagnosis of Chlamydia pneumoniae infection.
The diagnostic agent for Chlamydia pneumoniae infection according to claim 12 has the effect of the diagnostic agent according to claim 11 and can be immobilized on a carrier or the like because the amino acid sequence of the fusion protein used as an antigen is short. The number of antigenic peptides can be increased and the sensitivity is high.
The diagnostic agent for Chlamydia pneumoniae infection according to claim 13 has the effect of the diagnostic agent according to claim 11, and further, a fusion protein used as an antigen can form a structure that is difficult to be degraded by proteolytic enzymes. Excellent stability and high reliability of diagnostic results.
[0094]
The diagnostic agent for Chlamydia pneumoniae infection according to claim 14 exhibits the effect of the diagnostic agent according to claim 11, and further, since the fusion protein used as an antigen has the entire antigen polypeptide specific to Chlamydia pneumoniae, It is extremely suitable for antibody testing and accurate diagnosis of Chlamydia pneumoniae infection.
The diagnostic agent for Chlamydia pneumoniae infection according to claim 15 has the effect of the diagnostic agent according to claim 11, and further, since the fusion protein used as an antigen has an antigen part specific to Chlamydia pneumoniae, antibody testing And is extremely suitable for accurate diagnosis of Chlamydia pneumoniae infection.
[0095]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 160
Sequence type: amino acid
Sequence type: Peptide

[0096]
SEQ ID NO: 2
Sequence length: 488
Sequence type: amino acid
Sequence type: Peptide

[0097]
SEQ ID NO: 3
Array length: 649
Sequence type: amino acid
Sequence type: Peptide

[0098]
SEQ ID NO: 4
Sequence length: 432
Sequence type: amino acid
Sequence type: Peptide

[0099]
SEQ ID NO: 5
Sequence length: 1947
Sequence type: Nucleic acid
Number of strands: double strand
Sequence type: Other nucleic acids Synthetic DNA

[0100]
SEQ ID NO: 6
Sequence length: 1296
Sequence type: Nucleic acid
Number of strands: double strand
Sequence type: Other nucleic acids Synthetic DNA

[0101]
SEQ ID NO: 7
Sequence length: 1048
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: Genomic DNA
origin
Name of organism: C. pneumoniae
Stock Name: YK-41
Direct origin
Clone: 53-3S
Sequence features
Characteristic symbol: CDS
Location: 236..1012
How the features were determined: P

[0102]
SEQ ID NO: 8
Sequence length: 29
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
GATCCTGATG TCTATTTCAT CTTCTTCAG 29
[0103]
SEQ ID NO: 9
Sequence length: 28
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
GTCCTGAAGA AGATGAAATA GACATCAG 28
[0104]
SEQ ID NO: 10
Array length: 30
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
AATTGCCATG GGGGCCCTTA ATTAATTAAC 30
[0105]
SEQ ID NO: 11
Array length: 30
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
TCGAGTTAAT TAATTAAGGG CCCCCATGGC 30
[0106]
SEQ ID NO: 12
Sequence length: 5438
Sequence type: Nucleic acid
Number of strands: double strand
Sequence type: Other nucleic acid Plasmid
Array
ATCGATGTTA ACAGATCTAA GCTTAACTAA CTAACTCCGG AAAAGGAGGA ACTTCCATGA 60
TCAGTCTGAT TGCGGCGTTA GCGGTAGATC GCGTTATCGG CATGGAAAAC GCCATGCCGT 120
GGAACCTGCC TGCCGATCTC GCCTGGTTTA AACGCAACAC CTTAAATAAA CCCGTGATTA 180
TGGGCCGCCA TACCTGGGAA TCAATCGGTC GTCCGTTGCC AGGACGCAAA AATATTATCC 240
TCAGCAGTCA ACCGGGTACG GACGATCGCG TAACGTGGGT GAAGTCGGTG GATGAAGCCA 300
TCGCGGCGTG TGGTGACGTA CCAGAAATCA TGGTGATTGG CGGCGGTCGC GTTTATGAAC 360
AGTTCTTGCC AAAAGCGCAA AAACTGTATC TGACGCATAT CGACGCAGAA GTGGAAGGCG 420
ACACCCATTT CCCGGATTAC GAGCCGGATG ACTGGGAATC GGTATTCAGC GAATTCCACG 480
ATGCTGATGC GCAGAACTCT CACAGCTATG AGTTCGAAAT TCTGGAGCGG CGGATCCTGA 540
TGTCTATTTC ATCTTCTTCA GGACCTGACA ATCAAAAAAA TATCATGTCT CAAGTTCTGA 600
CATCGACACC CCAGGGCGTG CCCCAACAAG ATAAGCTGTC TGGCAACGAA ACGAAGCAAA 660
TACAGCAAAC ACGTCAGGGT AAAAACACTG AGATGGAAAG CGATGCCACT ATTGCTGGTG 720
CTTCTGGAAA AGACAAAACT TCCTCGACTA CAAAAACAGA AACAGCTCCA CAACAGGGAG 780
TTGCTGCTGG GAAAGAATCC TCAGAAAGTC AAAAGGCAGG TGCTGATACT GGAGTATCAG 840
GAGCGGCTGC TACTACAGCA TCAAATACTG CAACAAAAAT TGCTATGCAG ACCTCTATTG 900
AAGAGGCGAG CAAAAGTATG GAGTCTACCT TAGAGTCACT TCAAAGCCTC AGTGCCGCGC 960
AAATGAAAGA AGTCGAAGCG GTTGTTGTTG CTGCCCTCTC AGGGAAAAGT TCGGGTTCCG 1020
CAAAATTGGA AACACCTGAG CTCCCCAAGC CCGGGGTGAC ACCAAGATCA GAGGTTATCG 1080
AAATCGGACT CGCGCTTGCT AAAGCAATTC AGACATTGGG AGAAGCCACA AAATCTGCCT 1140
TATCTAACTA TGCAAGTACA CAAGCACAAG CAGACCAAAC AAATAAACTA GGTCTAGAAA 1200
AGCAAGCGAT AAAAATCGAT AAAGAACGAG AAGAATACCA AGAGATGAAG GCTGCCGAAC 1260
AGAAGTCTAA AGATCTCGAA GGAACAATGG ATACTGTCAA TACTGTGATG ATCGCGAAGG 1320
GGTTCGAATT GCCATGGGGG CCCTTAATTA ATTAACTCGA GAGATCCAGA TCTAATCGAT 1380
GATCCTCTAC GCCGGACGCA TCGTGGCCGG CATCACCGGC GCCACAGGTG CGGTTGCTGG 1440
CGCCTATATC GCCGACATCA CCGATGGGGA AGATCGGGCT CGCCACTTCG GGCTCATGAG 1500
CGCTTGTTTC GGCGTGGGTA TGGTGGCAGG CCCGTGGCCG GGGGACTGTT GGGCGCCATC 1560
TCCTTGCATG CACCATTCCT TGCGGCGGCG GTGCTCAACG GCCTCAACCT ACTACTGGGC 1620
TGCTTCCTAA TGCAGGAGTC GCATAAGGGA GAGCGTCGAC CGATGCCCTT GAGAGCCTTC 1680
AACCCAGTCA GCTCCTTCCG GTGGGCGCGG GGCATGACTA TCGTCGCCGC ACTTATGACT 1740
GTCTTCTTTA TCATGCAACT CGTAGGACAG GTGCCGGCAG CGCTCTGGGT CATTTTCGGC 1800
GAGGACCGCT TTCGCTGGAG CGCGACGATG ATCGGCCTGT CGCTTGCGGT ATTCGGAATC 1860
TTGCACGCCC TCGCTCAAGC CTTCGTCACT GGTCCCGCCA CCAAACGTTT CGGCGAGAAG 1920
CAGGCCATTA TCGCCGGCAT GGCGGCCGAC GCGCTGGGCT ACGTCTTGCT GGCGTTCGCG 1980
ACGCGAGGCT GGATGGCCTT CCCCATTATG ATTCTTCTCG CTTCCGGCGG CATCGGGATG 2040
CCCGCGTTGC AGGCCATGCT GTCCAGGCAG GTAGATGACG ACCATCAGGG ACAGCTTCAA 2100
GGATCGCTCG CGGCTCTTAC CAGCCTAACT TCGATCACTG GACCGCTGAT CGTCACGGCG 2160
ATTTATGCCG CCTCGGCGAG CACATGGAAC GGGTTGGCAT GGATTGTAGG CGCCGCCCTA 2220
TACCTTGTCT GCCTCCCCGC GTTGCGTCGC GGTGCATGGA GCCGGGCCAC CTCGACCTGA 2280
ATGGAAGCCG GCGGCACCTC GCTAACGGAT TCACCACTCC AAGAATTGGA GCCAATCAAT 2340
TCTTGCGGAG AACTGTGAAT GCGCAAACCA ACCCTTGGCA GAACATATCC ATCGCGTCCG 2400
CCATCTCCAG CAGCCGCACG CGGCGCATCT CGGGCAGCGT TGGGTCCTGG CCACGGGTGC 2460
GCATGATCGT GCTCCTGTCG TTGAGGACCC GGCTAGGCTG GCGGGGTTGC CTTACTGGTT 2520
AGCAGAATGA ATCACCGATA CGCGAGCGAA CGTGAAGCGA CTGCTGCTGC AAAACGTCTG 2580
CGACCTGAGC AACAACATGA ATGGTCTTCG GTTTCCGTGT TTCGTAAAGT CTGGAAACGC 2640
GGAAGTCAGC GCCCTGCACC ATTATGTTCC GGATCTGCAT CGCAGGATGC TGCTGGCTAC 2700
CCTGTGGAAC ACCTACATCT GTATTAACGA AGCGCTGGCA TTGACCCTGA GTGATTTTTC 2760
TCTGGTCCCG CCGCATCCAT ACCGCCAGTT GTTTACCCTC ACAACGTTCC AGTAACCGGG 2820
CATGTTCATC ATCAGTAACC CGTATCGTGA GCATCCTCTC TCGTTTCATC GGTATCATTA 2880
CCCCCATGAA CAGAAATTCC CCCTTACACG GAGGCATCAA GTGACCAAAC AGGAAAAAAC 2940
CGCCCTTAAC ATGGCCCGCT TTATCAGAAG CCAGACATTA ACGCTTCTGG AGAAACTCAA 3000
CGAGCTGGAC GCGGATGAAC AGGCAGACAT CTGTGAATCG CTTCACGACC ACGCTGATGA 3060
GCTTTACCGC AGCTGCCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA 3120
GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA 3180
GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC ATGACCCAGT CACGTAGCGA 3240
TAGCGGAGTG TATACTGGCT TAACTATGCG GCATCAGAGC AGATTGTACT GAGAGTGCAC 3300
CATATGCGGT GTGAAATACC GCACAGATGC GTAAGGAGAA AATACCGCAT CAGGCGCTCT 3360
TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA 3420
GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC 3480
ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT 3540
TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG 3600
CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC 3660
TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC 3720
GTGGCGCTTT CTCAATGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC 3780
AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC 3840
TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT 3900
AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT 3960
AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC 4020
TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT 4080
TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG 4140
ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC 4200
ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA 4260
TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG 4320
GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG 4380
TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA 4440
GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG 4500
CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA 4560
GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTGCAGGC 4620
ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA 4680
AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG 4740
ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT 4800
AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC 4860
AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAACACGG 4920
GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG 4980
GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT 5040
GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA 5100
GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA 5160
CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC 5220
ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA 5280
GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT TAACCTATAA AAATAGGCGT 5340
ATCACGAGGC CCTTTCGTCT TCAAGAATTA ATTGTTATCC GCTCACAATT AATTCTTGAC 5400
AATTAGTTAA CTATTTGTTA TAATGTATTC ATAAGCTT 5438
[0107]
SEQ ID NO: 13
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
GCTGCCGAAC AGAAGTCTAA 20
[0108]
SEQ ID NO: 14
Sequence length: 20
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
CTCGAAGGAA CAATGGATAC 20
[0109]
SEQ ID NO: 15
Sequence length: 23
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
GTACATATTG TCGTTAGAAC GCG 23
[0110]
SEQ ID NO: 16
Sequence length: 23
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
TAATACGACT CACTATAGGG AGA 23
[0111]
SEQ ID NO: 17
Sequence length: 28
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
GCGGATCCTG ATGTCTATTT CATCTTCT 28
[0112]
SEQ ID NO: 18
Array length: 30
Sequence type: Nucleic acid
Number of chains: single chain
Sequence type: Other nucleic acids Synthetic DNA
Array
ATCTCGAGTT TTATGCTGCT GCGCCAGCGA 30

Claims (15)

A gene set in which polypeptide A comprising a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 2 is bound to the peptide represented by SEQ ID NO: 1 directly or via an intervening amino acid sequence A method for measuring an anti-Chlamydia pneumoniae antibody, characterized by using a dihydrofolate reductase-Chlamydia pneumoniae antigen fusion protein produced by a replacement method as an antigen.   The method for measuring an anti-Chlamydia pneumoniae antibody according to claim 1, wherein the polypeptide A is a polypeptide lacking an amino acid from the peptide represented by SEQ ID NO: 2.   The polypeptide A is a polypeptide in which an amino acid in the peptide represented by SEQ ID NO: 2 is substituted with another amino acid or an amino acid is inserted into the peptide represented by SEQ ID NO: 2. A method for measuring the anti-Chlamydia pneumoniae antibody described.   The method for measuring an anti-Chlamydia pneumoniae antibody according to claim 1, wherein the fusion protein is a peptide represented by SEQ ID NO: 3.   The method for measuring an anti-Chlamydia pneumoniae antibody according to claim 1, wherein the fusion protein is a peptide represented by SEQ ID NO: 4. A gene set in which polypeptide A comprising a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 2 is bound to the peptide represented by SEQ ID NO: 1 directly or via an intervening amino acid sequence A reagent for measurement of an anti-Chlamydia pneumoniae antibody comprising, as an antigen, a dihydrofolate reductase-Chlamydia pneumoniae antigen fusion protein produced using a replacement method .   The reagent for measuring an anti-Chlamydia pneumoniae antibody according to claim 6, wherein the polypeptide A is a polypeptide lacking an amino acid from the peptide represented by SEQ ID NO: 2.   The polypeptide A is a polypeptide in which an amino acid in the peptide represented by SEQ ID NO: 2 is substituted with another amino acid or an amino acid is inserted into the peptide represented by SEQ ID NO: 2. The reagent for a measurement of anti-Chlamydia pneumoniae antibody as described.   The reagent for measuring an anti-Chlamydia pneumoniae antibody according to claim 6, wherein the fusion protein is a peptide represented by SEQ ID NO: 3.   The reagent for measuring an anti-Chlamydia pneumoniae antibody according to claim 6, wherein the fusion protein is the peptide represented by SEQ ID NO: 4. A gene set in which polypeptide A comprising a sequence consisting of at least 5 consecutive amino acids in the peptide represented by SEQ ID NO: 2 is bound to the peptide represented by SEQ ID NO: 1 directly or via an intervening amino acid sequence A diagnostic agent for Chlamydia pneumoniae infection, comprising as an active ingredient a dihydrofolate reductase-Chlamydia pneumoniae antigen fusion protein produced using a replacement method .   The diagnostic agent for Chlamydia pneumoniae infection according to claim 11, wherein the polypeptide A is a polypeptide lacking an amino acid from the peptide represented by SEQ ID NO: 2.   The polypeptide A is a polypeptide in which an amino acid in the peptide represented by SEQ ID NO: 2 is substituted with another amino acid or an amino acid is inserted into the peptide represented by SEQ ID NO: 2. The diagnostic agent of Chlamydia pneumoniae infection as described.   The diagnostic agent for Chlamydia pneumoniae infection according to claim 11, wherein the fusion protein is a peptide represented by SEQ ID NO: 3.   The diagnostic agent for Chlamydia pneumoniae infection according to claim 11, wherein the fusion protein is the peptide represented by SEQ ID NO: 4.