JP3582663B2 - Gene clone expressing chicken leukocytozone immunogenic protein and transgenic vaccine against chicken leukocytosonosis - Google Patents

Description

【化１２】

【化１３】

【００２０】
本発明において使用可能なベクターとしては、大腸菌発現ベクター、大腸菌フアージ系ベクター、大腸菌−培養細胞シヤトルベクター、ウイルスベクター等であって、宿主細胞、特に大腸菌内で蛋白質を発現できるものであれば特に限定されない。本発明における発現蛋白質によりロイコチトゾーン症のワクチンを調製する場合、特に精製の操作なしに感染細胞の抽出液をそのまま使用しても防御効果が認められる。濃縮は例えば大腸菌体内での発現の場合であるが、培養液の遠心沈澱菌体の超音波処理時の浮遊液量で、または超音波抽出液の硫安濃縮沈澱の溶解液量でその度合が加減できる。鶏にはアジュバンドとしては例えば水酸化アルミニウムゲルと混合して、公知の方法により免疫ができる。
【００２１】
本発明に用いられる原材料と好ましい組合せについて以下に述べる。
（１）組換え微生物ＴＢ１／ｐＭＡＬ−ｃＲ１−Ｒ７
マスターセルストック及びワーキングシードストックとして−２０℃で保存されていて、ワクチンを作出する出発材料となる保存株である。
（２）アンピシリン
宿主大腸菌ＴＢ１株に移入された発現ベクターｐＭＡＬ−ｃＲ１−Ｒ７はアンピシリン耐性遺伝子を有し、これが宿主菌の培養に一つの選択性を与える。
（３）Ｌ−ブロス寒天培地
ワーキングシードから一次元培養を行うための培地で、１％トリプトン、０．５％イーストエキストラクト及び１％塩化ナトリウムを主成分とし、１％細菌用寒天末および１００μｇ／ｍｌアンピシリンを含む。
【００２２】
（４）Ｌ−ブロス培地、ｐＨ７．０
一次元培養から二次元培養を行うための培地で、上記Ｌ−ブロス寒天培地で寒天末を除いたもの。
（５）Ｌ−ブロス発現培養培地、ｐＨ７．０
組換え微生物の発現培養を行うための培地で、上記のＬ−ブロス培地でさらに０．２％グルコースを含む。
（６）イソプロピル−β−Ｄ−チオガラクトピラノシド
発現培養時の対数増殖期に培養液に添加する発現誘発剤。
（７）リシスバッファー、ｐＨ７．０
培養菌体を超音波処理する際に、菌体を浮遊させる緩衝液で、１０ｍＭリン酸塩、３０ｍＭ塩化ナトリウム、０．２５％ツイーン２０、１０ｍＭ ２−メルカプトエタノール、１０ｍＭＥＤＴＡ、及び１０ｍＭＥＧＴＡを含む。
【００２３】
（８）飽和硫酸アンモニウム
菌体の超音波抽出液の有効成分を沈澱させる。
（９）生理緩衝食塩水（ＰＢＳ）、ｐＨ７．０
飽和硫酸アンモニウムによる有効成分の沈澱物を溶解させる。
（１０）ホルマリン
ワクチンの不活化及び防腐を目的として終濃度０．２％に添加する。
（１１）水酸化アルミニウム
ワクチンのアジュバントとして、終濃度５００μｇＡ１／ｍｌに添加する。
【００２４】
本発明に用いられる原材料の一般的使用量、好ましい使用量の範囲
（１）組換え微生物ＴＢ１／ｐＭＡＬ−ｃＲ１−Ｒ７のワーキングシードストツクス：１回にエーゼルのループフル量または数μ１
（２）アンピシリン：１００μｇ／ｍｌ
（３）Ｌ−ブロス寒天培地、ｐＨ７．０：１回に２０ｍｌ／プレートの１プレート
以下は組換え微生物１００ｌ 培養トその処理に対する使用量である。
（４）Ｌ−ブロス培地、ｐＨ７．０：２ｌ
（５）Ｌ−ブロス発現培養培地、ｐＨ７．０：１００ｌ
（６）イソプロピル−β−Ｄ−チオガラクトピラノシド：１Ｍ溶液、１００ｍｌ
【００２５】
（７）リシスバッファー、ｐＨ７．０：６．７ｌ （発現培養液の約１／１５量）
（８）飽和硫酸アンモニウム：６．７ｌ （リシスバッファーと同量）
（９）ＰＢＳ、ｐＨ７．０：５ｌ
（１０）ホルマリン：１０％、１００ｍｌ×２（下記の水酸化アルミニウムゲル添加後の添加量を含む）
（１１）水酸化アルミニウムゲル：１０００μｇＡ１／ｍｌ、５ｌ
【００２６】
【作用及び効果】
（１）作用
本発明によるワクチンは形質転換微生物による生産物であることから、次の点で鶏で生産される原虫抗原採取法より優れている。
１）安全性：本ワクチンに使用される組換え微生物は病原性、有毒物質産生性、及び伝達性はないことから本ワクチンもそのような性質はない。また本ワクチンは組換え微生物の超音波処理による抽出液で、しかもホルマリンで不活化したものであるので、感染性のおそれもない。
２）経済性：原虫採取のためのヌカカ、鶏及びそれらに要する飼育設備、施設、人手などの全てを省くことができるので、経済性に優れている。
３）生産性：組換え微生物の宿主菌は大腸菌であるため、容易に大量培養が可能となり、また発現蛋白質の精製の操作を必要としないので、生産性が高い。
【００２７】
（２）効果
１）本ワクチンは実施例４に記載したように免疫後、スポロゾイトを鶏あたり１３０００〜１５０００匹を静脈内に接種して実験感染させても、非免疫群に比較して明らかに原虫の発育を抑えることから、本目的を適えるものである。
２）前記の安全性の点から環境への病原体汚染の問題はない。
３）発現培養による各標品のＥＬＩＳＡ抗原価はほぼ一定していることから、抗原の量的変動が少なく、均一なワクチンを提供できる。
【００２８】
【実施例】
以下に本発明の実施例を具体的に説明するが、本発明はこれにより制限されるものでないことはいうまでもまない。
実施例
ロイコチトゾーン・カウレリー第２代シゾントの免疫原性蛋白質遺伝子のクローニング、ワクチンクローンの選定、及び遺伝子組換えワクチンの感染防
御機能試験
（１）ロイコチトゾーン・カウレリー第２代シゾント由来ｃＤＮＡライブラリー
の調製
ヌカカより調製したロイコチトゾーン・カウレリーのスポロゾイトを約５週令の鶏１羽当たり約１００００匹静脈内に接種して実験感染させた。感染後第２代シゾント（２ＧＳ）が出現する時期の１３日目に鶏を解剖し、胸腺、肺、肝、腎、脾、Ｆ嚢の各臓器を採取し、それらの臓器乳剤から２ＧＳを分離、精製した。
【００２９】
これよりＲＮＡをグアニジウムイソチオシアネート法により抽出し、塩化リチウムによる分別沈澱を行い、さらにエタノール沈澱を行って総ＲＮＡを得た後、ｍＲＮＡをオリゴテックス−ｄＴ３０（宝酒造社製）を用いて、２回精製を繰り返して得た。次に、市販のキット（アマーシャム社製）を用いｍＲＮＡを鋳型として、オリゴ（ｄＴ）ヌクレオチドまたはランダムヘキサヌクレオチドをプライマーとして逆転写酵素でギュブラーとホフマンの方法に準じてｃＤＮＡを合成した。
【００３０】
得られた１本鎖ｃＤＮＡにＲＮａｓｅＨとＤＮＡポリメラーゼとを作用させて２本鎖ｃＤＮＡにした後、ＥｃｏＲＩアダプター（宝酒造社製）を付加したものをラムダファージλｇｔ１１（アマーシャム社製）の両アームとライゲーションさせ、インビトロパッケージングキット（ストラタジーン社製）を用いてラムダファージのパッケージングを行ってｃＤＮＡライブラリーを調製した。
ｃＤＮＡライブラリーを調製するにあたり、ＥｃｏＲＩアダプター付加ｃＤＮＡをアガロース電気泳動で４つにサイズ分画した。これらについてのライゲーション操作後にインビトロパッケージングを行った。従って、ｃＤＮＡライブラリーは２種のプライマーのｃＤＮＡライブラリーは２種のプライマーのｃＤＮＡについてそれぞれ４種が調製された。
【００３１】
（２）ｃＤＮＡライブラリーのプラークイムノスクリーニング及びクローンの選 定
２種のプライマーに基づいた各ｃＤＮＡライブラリーで、その調製前のサイズ分画に由来する各サイズの小さい方の２種（＜５００ｂｐ）は省き、大きい方の２種（＞５００ｂｐ）について、それぞれ大腸菌Ｙ１０９０株に感染させ、１００μｇ／ｍｌのアンピシリンを含むＬ−ブロス寒天培地上で常法にしたがってプラークの生成を行った。常法にしたがって、ｌａｃプロモーターの誘発剤であるイソプロピル−β−Ｄ−チオガラクトピラノシド（ＩＰＴＧ）を含浸させたニトロセルロースフィルターに写し取り、イムノスクリーニングを行った。スクリーニングに用いた一次抗体には、２ＧＳ抗原の６７ｋＤａ付近を認識するＭ５と１００〜２００ｋＤａを認識するＲ１の２種の単クローン抗体の等量混合物を用いた。２次抗体にはＨＲＰ標識家兎抗マウスＩｇＧ＋ＩｇＭ＋ＩｇＡを、発色反応の基質にはジアミノベンチジンを用いた。
【００３２】
単クローン抗体は、予めそれぞれの単クローン抗体結合アフィニティーカラムで精製した各２ＧＳ抗原の混合物での試験であるが、感染防御性が認められ、本スクリーニングに適していると判断されたものである。スクリーニングの見落としを防止することからも、両者の混合物を用いて行った。
尚、両者共それによって認識される抗原は分子量的に単一ではなく、それぞれ上述したようなある範囲のものが認識されることから、本原虫抗原は同一エピトープの繰り返し構造を有しているものと推察される。
【００３３】
クローニングの結果、オリゴ（ｄＴ）ヌクレオチドプライマーに基づいたｃＤＮＡの場合で３３種（Ｔ番号を付す）の、ランダムヘキサヌクレオチドプライマーに基づいたｃＤＮＡの場合で４４種（Ｒ番号を付す）の組換えＤＮＡが得られた。それらを再クリーニングした後、クローンの選定を行った。
【００３４】
即ち、再スクリーニングした際にも発色が比較的強い陽性反応を示したファージを増殖後、大腸菌Ｙ１０８９株に感染させて溶原菌を調製した後、誘発剤にＩＰＴＧを用いた発現培養を行い、細胞ライセートを調製して同単クローン抗体の混合物を用いてイムノドットブロットテストを行って抗原の発現を再確認すると共に、一方、陽性ファージを増殖、精製後ＥｃｏＲＩ−切断フラグメント生成の確認を行い、その結果Ｒ１ａ、Ｒ１ｂ、Ｒ４ａ、Ｒ４ｂ、Ｒ６、Ｒ７、Ｒ１１、Ｒ１３、Ｒ１５、Ｒ１８、Ｒ２９、Ｒ３５、Ｒ４２、Ｔ３、Ｔ４、Ｔ９、Ｔ１０の１７種を免疫原性蛋白質遺伝子の有力なクローンとして選定した。以上の工程の要約を図１、図２及び図３に示した。
【００３５】
このようにして得られたクローンが、実際に免疫原性を賦与し得るものかをみるために、この段階でＲ１５、Ｔ１０の任意の２種の細胞ライセートについて、アジュバントとして水酸化アルミニウムゲルを５００μｇＡｌ／ｍｌに添加して感染防御試験を試みた。感染防御試験の方法と効果判定の方法の概略については実施例の（４）に述べるが、Ｒ１５及びＴ１０λｇｔ１１クローンの発現蛋白質の感染防御試験の結果は表１に示すように未接種対照群も含めて全例に発症、死亡が認められなかったが、免疫鶏にスポロゾイト１９５００匹／羽で攻撃を行った後、１３日目に血清中に出現する原虫の発育過程に由来する血清可溶性抗原（ＳＳＡ）の量的値（ＳＳＡ価）では、その値の低い羽数が免疫群は未接種対照群を上回ったことから、Ｒ１５、Ｔ１０には弱いながらも免疫性が認められたと判断した。
【００３６】
また攻撃直前の血清の２ＧＳ抗原に対するワクチン抗体価（ＥＬＩＳＡ抗体価）も免疫群では全例未接種対照群に比較して上昇していることが認められた。このことから本スクリーニングによって得られたクローンは本目的に適うものと判断した。
【００３７】
【表１】

【００３８】
（３）発現ベクターｐＭＡＬ−ｃＲＩサブクローン及びワクチンクローンの選定ワクチン実用化の発現培養のために、上記１７種のクローンの当該遺伝子をそれぞれ発現ベクターｐＭＡＬ−ｃＲＩ（バイオラブス社製）に挿入し、上述と同様のイムノスクリーニングでｐＭＡＬ−ｃＲＩサブクローンを得て、大腸菌ＴＢ１株に移入した。これらのサブクローンについて２種の単クローン抗体それぞれに対するＥＬＩＳＡ抗原価測定とサザーンハイブリダイゼーションによりクローンの群別を試みてワクチンクローンの選定を行った。ＥＬＩＳＡには発現培養を行って調製した試料を、サザーンハイブリダイゼーションには、一方で当該遺伝子をｐＵＣ１９にもサブクローンして調製したプラスミドを用いた。
【００３９】
製剤の作製方法でもある発現培養による試料の調製方法は次の通りである。宿主菌のＬ−ブロス液体培地を用いた３７℃での振とう培養で対数増殖期にＩＰＴＧを終濃度１ｍＭに添加して、さらに３７℃で４〜６時間培養して蛋白質をマルトース結合蛋白質との融合蛋白質として発現させた後、遠心分離して得られた菌体をリシスバッファーに浮遊、超音波処理し、再び遠心分離して超音波抽出液を得た。さらにこの抽出液を飽和硫酸アンモニウムで処理して得られた沈澱をＰＢＳに溶解し、ホルマリンを終濃度０．２％に添加し、４℃で１晩以上静置後試料とした。
【００４０】
ＥＬＩＳＡ抗原価の測定結果では、単クローン抗体Ｍ５及びＲ１のいずれかと明らかな反応性を示したクローンは１７種の中８種であり、その中、単クローン抗体Ｍ５反応群はＲ１、Ｒ４ａ、Ｒ１３及びＴ３で、単クローン抗体Ｒ１反応群はＲ７、Ｒ１５、Ｒ３５及びＴ１０でいずれも４種ずつであった。なお、単クローン抗体Ｒ１と強い反応性を示したＲ７、Ｒ１５は、培養によっては単クローン抗体Ｍ５とも反応性を示した。
【００４１】
ｐＭＡＬ−ｃＲＩサブクローンの発現蛋白質（粗物質）のＥＬＩＳＡ抗体価及びサザーンハイブリダイゼーションによる群別とクローンのサイズ、並びにＥＬＩＳＡ抗原価：単クローン抗体Ｍ５及びＲ１それぞれに対する反応性、サザーンハイブリダイゼーション：プローブＲ４ａ、Ｒ７及びＲ１５に対する反応性の測定結果を表２に示した。
【００４２】
一方、プローブとしてＲ４ａ、Ｒ７及びＲ１５の各プラスミド由来精製フラグメントを用いたサザーンハイブリダイゼーションの結果では図３に示すようにプローブＲ４に対してＲ４ａ、Ｔ３が、プローブＲ７とＲ１５に対してはいずれの場合もＲ７、Ｒ１５、Ｒ３５及びＴ１０が反応性を示した。これらについて各クローンのＤＮＡサイズ（近似値）とともに表２に示した。
【００４３】
【表２】

【００４４】
サザーンハイブリダイゼーションで、プローブＲ４ａと反応したものはＲ４ａとＴ３で、ＥＬＩＳＡ抗原価による単クローン抗体Ｍ５反応群としては同一群であるＲ１ａ、Ｒ１３との反応性は認められなかったが、ＥＬＩＳＡ抗原価による２種の単クローン抗体を認識するクローンの群別と、サザーンハイブリダイゼーションによる遺伝子の相互類似性を示すクローンの群別とは一致するものと判断した。
【００４５】
そこで、単クローン抗体Ｒ１認識群とＭ５認識群の各４種のクローンの発現試料の混合物を用いた感染防御試験の比較を試みた。その結果は表３に示した。スポロゾイト攻撃時の血清の２ＧＳ抗原に対するＥＬＩＳＡ抗体価では明らかにＲ１群はＭ５群を上回り、また攻撃後１３日目のＳＳＡの抑制程度もＲ１群はＰＢＳ対照群を有意に上回っていた。さらに８種の個々のクローンを任意に選んで行った感染防御成績からは、単クローン抗体Ｍ５と反応する代表的なクローンＲ４ａによる免疫抗体は、ＥＬＩＳＡで同じＲ４ａの抗原とは反応するが２ＧＳ抗原とは常に反応性を示さず、これに対して単クローン抗体Ｒ１と反応するクローンによる免疫抗体は、同法で同じ抗原ともまた２ＧＳ抗原とも反応性を示すことがわかった。
【００４６】
一方、また８種のウエスタンブロットの成績からも、単クローン抗体Ｍ５は、当該遺伝子を挿入しないベクターを移入した宿主菌由来の抽出液（発現の陰性対照）の数多くの蛋白質をも認識するが、これに対し単クローン抗体Ｒ１はそのような現象を示さず、常に発現によって生じた蛋白質を主として認識するものであることがわかった。
【００４７】
【表３】

【００４８】
以上のことから、ワクチンクローンには２ＧＳ抗原に対して特異性を示すＲ１単クローン抗体認識クローンから選定することが適切であると判断した。これらのクローンについてさらに感染防御試験で検討した結果、Ｒ７、Ｒ１５の両クローンがワクチンクローンの候補に挙げられたが、効果を示す回数が上回ったＲ７が最終的にワクチンクローンとして決定された。このようにして得られたＲ７クローンの組換え微生物をＴＢ１／ｐＭＡＬ−ｃＲ１−Ｒ７と命名し、これを発現させて作製したワクチンをロイコチトゾーン症遺伝子組換えワクチン（Ｒ７ワクチンと略称）した。なお、本組換え微生物ＴＢ１／ｐＭＡＬ−ｃＲ１−Ｒ７は工業技術院生命工学工業技術研究所に平成６年３月２２日、受託番号ＦＥＲＭ Ｐ−１４２４０として寄託されている。
【００４９】
次に、本微生物ＴＢ１／ｐＭＡＬ−ｃＲ１−Ｒ７について以下の成績を説明する。
ｐＭＡＬ−ｃＲ１−Ｒ７切断Ｒ７フラグメントの電気泳動像：
組換え微生物ＴＢ１／ｐＭＡＬ−ｃＲ１−Ｒ７の増殖培養菌から精製したｐＭＡＬ−ｃＲ１−Ｒ７プラスミドとこれのＥｃｏＲＩ−切断ＤＮＡについて、１．２％アガロースゲル電気泳動を行い、エチジウムブロマイドで染色した。図４は当該組換え微生物からｐＭＡＬ−ｃＲ１−Ｒ７プラスミドが得られ、このプラスミドからＥｃｏＲＩ−切断により、約６．１ｋｂｐのベクターと約２．０ｋｂｐのＲ７フラグメントが生じることを示す。
【００５０】
本微生物ＴＢ１／ｐＭＡＬ−ｃＲ１−Ｒ７の発現材料の電気泳動像及びウエス
タンブロット像：
発現培養液の液量に対し１／１０量の菌体浮遊液の超音波破砕抽出液を、さらに飽和硫酸アンモニウムで処理して１／４０量とした発現材料に、等量の泳動用試料緩衝液を加え、これの２０μｌをＳＤＳ−ポリアクリルアミドゲル（８％濃度のゲル）にアプライして泳動を行った（ＳＤＳ−ＰＡＧＥ）。ウエスタンブロットの１次抗体には単クローン抗体Ｒ１を用いた。このときのＳＤＳ−ＰＡＧＥ像を図６（ａ）、（ｂ）に、ウエスタンブロット像を図７のそれぞれ示した。
【００５１】
図６（ａ）、（ｂ）に示すように発現材料では２００ｋＤａ近くに２本のバンドが出現したが、一方、発現の誘発をかけない非発現材料ではそれらのバンドはみられなかった。また、図７に示すように発現材料ではそれらのバンドに反応性がみられたが、一方、非発現材料では反応性はみられなかった。なお、発現材料の２０倍希釈での反応性は２本のバンドの中、一方だけになったとみられる。
【００５２】
このように２００ｋＤａ近くにバンドがみられる材料は、後述の単クローン抗体Ｒ１との反応でＥＬＩＳＡ抗原価が示されるが、さらにそれらの関連性をみるために、発現材料についてセファクリルＳ２００によるカラムクロマトグラフイー行い、その画分Ｆ１、Ｆ２及びＦ３のカラムアプライ量に対して各４倍濃縮材料について、同様にしてＳＤＳ−ＰＡＧＥ像およびウエスタンブロット像を調べた。このときのＳＤＳ−ＰＡＧＥ像およびウエスタンブロット像をそれぞれ図８及び図９に示した。
【００５３】
ＳＤＳ−ＰＡＧＥで２００ｋＤａ近くに２本のバンドがＦ１では、やはりウエスタンブロット反応性がみられるが、それらのバンドがみられないＦ２およびＦ３では反応性はみられなかった。なお、Ｆ１の２０倍希釈での反応性はやはり２本のバンドの中、一方だけになったとみられる。また、これらの画分についてのＥＬＩＳＡ抗原価はＦ１では認められたが、Ｆ２には認められなかった。
【００５４】
発現材料及びその濾過画分のＥＬＩＳＡ抗原価（１００倍に希釈した単クローン抗体Ｒ１を用いたときのＥＬＩＳＡ抗体価）はつぎの通りである。

【００５５】
このように、発現材料ではＳＤＳ−ＰＡＧＥにおいて２００ｋＤａ近くにバンドが出現し、それらがウエスタンブロット反応を示すのに対し、同様の非発現材料ではそれらのバンドは出現せず、ウエスタンブロットでも反応性がみられないこと、またＥＬＩＳＡ抗原価はそれらのバンドが出現する材料にのみ認められることから、それらの物質が発現融合蛋白質と判断された。Ｒ７遺伝子の塩基配列は以下の通りで、その両末端の下線部分はＥｃｏＲＩアダプター領域を示す。
【００５６】
【化１４】

【化１５】

【化１６】

【００５７】
本微生物ＴＢ１／ｐＭＡＬ−ｃＲ１−Ｒ７の発現融合蛋白質領域
融合蛋白質領域は、
【００５８】
【化１７】

【化１８】

【化１９】

【化２０】

で示すように、マルトース結合蛋白質遺伝子の最初の開始コドンＡＴＧから１ａｃＺａ遺伝子の最後の終止コドンＴＧＡまでの３３９９ｂｐ（１１３２個のアミノ酸）である。その中に２０１９ｂｐのＲ７遺伝子がＥｃｏＲＩアダプターを介して挿入されている。ＥｃｏＲＩアダプター領域は破線で、Ｒ７領域は実線で示した。Ｒ７遺伝子の両末端のＥｃｏＲＩアダプターを含めた領域の蛋白質は６８９個のアミノ酸から成り、その組成は次の通りである。
【００５９】
Ａｌａ ３（０．４） Ｌｅｕ ２（０．３）
Ａｒｇ ２（０．３） Ｌｙｓ ６８（９．９）
Ａｓｎ ２５（３．６） Ｍｅｔ ５（０．７）
Ａｓｐ １２（１．７） Ｐｈｅ ２（０．３）
Ｃｙｓ １（０．１） Ｐｒｏ ５（０．７）
Ｇｌｎ １４（２．０） Ｓｅｒ ６（０．９）
Ｇｌｕ ３１１（４５．１） Ｔｈｒ ５４（７．８）
Ｇｌｙ ９（１．３） Ｔｒｐ ０（０．０）
Ｈｉｓ ６８（９．９ Ｔｙｒ ８（１．２）
Ｉｌｅ ３５（５．１） Ｖａｌ ５９（８．６）
Ｅｎｄ ０（０．０）
【００６０】
酸性アミノ酸 （Ａｓｐ＋Ｇｌｕ） ３２３（４６．９）
塩基性アミノ酸 （Ａｒｇ＋Ｌｙｓ） ７０（１０．２）
芳香族アミノ酸 （Ｐｈｅ＋Ｔｒｐ＋Ｔｙｒ） １０（１．５）
疏水性アミノ酸 （芳香族アミノ酸＋Ｉｌｅ ＋Ｌｅｕ ＋Ｍｅｔ ＋Ｖａｌ ） １１１ （１６．１）
分子量＝８４１４４
【００６１】
（４）本Ｒ７ワクチンの感染防御試験
本原虫症ワクチンの感染防御試験による効果判定には、免疫鶏にスポロゾイトで攻撃後、主として１３日目の血清の可溶性抗原（ＳＳＡ）価の測定、２１日目までの発症、生・死判定を含む臨床症状の観察、及び２１日目の血液中のガメトサイト数の測定等の評価の結果がその評価の対象となり、その他ワクチン抗体価（攻撃直前の血清抗体価）の測定及び攻撃後２１日目の血液中の赤血球数の測定等の結果も評価の参考とした。
【００６２】
ＳＳＡ価またはガメトサイト数の測定は、スポロゾイトで攻撃後２１日目を経ても発症、生・死では効果判定不能の場合にも評価の対象になるものである。ＳＳＡ価の測定は、攻撃スポロゾイトにより血清中に出現する原虫の発育過程に由来するＳＳＡ価の量を、感染耐過鶏血清を用いたゲル内沈降反応で調べ、陽性反応の程度が低いもの程、スポロゾイトの発育が抑えられたことになり、したがってその反応が少ない羽数が非免疫対照群の羽数に比較して多いもの程、防御効果が認められたことになる。同様にガメトサイト数の測定はやはりスポロゾイトがガメトサイトにまで発育するその数を測定するもので、血液中にその数が少ない羽数が非免疫対照群の羽数に比較して多いもの程、防御効果が認められたことになる。
【００６３】
ワクチンは前述の実施例の（３）に記載した通りに調製し、最終的に水酸化アルミニウムゲルを加えた時点でその量は培養液量に対して１／１０とした。以下３ロットの試作品についてそれぞれ試験−１、試験−２及び試験−３として行った。鶏は試験−１、試験−２及び試験−３ではそれぞれ３６日令、２３日令および２１日令のいずれもＳＰＦ鶏由来ひなを用い、ワクチンを左右大腿部筋肉内に０．５ｍｌずつ計１ｍｌを２週間隔で２回注射した。試験−３ではさらに１．５ｍｌずつの計３ｍｌでも試みた。ワクチン２回注射２週間後に血清を採取した後、攻撃はロイコ原虫静岡株のスポロゾイトを１羽あたり、試験−１では１３６００匹、試験−２では１２５００匹、試験−３では１５０００匹を静脈内接種して行った。
【００６４】
攻撃直前に採血し、攻撃後２１日間臨床症状の観察を行い、この間１３日目に採血し、また観察終了時に血液サンプルを採取した。さらに攻撃時及び試験終了時の体重を測定した。
それらによる試験−１、試験−２及び試験−３における主として、ワクチン抗体価（攻撃直前のＥＬＩＳＡによる血清抗体価）、攻撃後１３日目のＳＳＡ価、攻撃後２１日目までの臨床症状観察、試験終了時のガメトサイト数等及び増体重の結果をそれぞれ表４、表５及び表６に示した。
【００６５】
試験−１
【００６６】
【表４】

１）陽性を示した血清の最高希釈倍数
２）沈降線のみられた血清の最高希釈倍数
３）攻撃後日数
４）死亡のための検査できず
５）血液凝固のため検査できず
【００６７】
表４の結果から明らかなように、ＥＬＩＳＡ抗体価はワクチン群では＜１００〜１６００倍で、対照群の全例＜１００倍に比較して上昇が認められた。ＳＳＡ価は、ワクチン群では認められた抗原は２〜３２倍（平均１４．５倍）であったのに対し、対照群では８〜＞５１２倍（平均６４．０倍）の抗原が認められ、両者の間には明らかな差が認められた。
【００６８】
臨床症状観察結果では、ワクチン群では８例中３例が発症、その中２例が死亡したのに対し、対照群では８例中全例が発症し、その中４例が死亡した。ガメトサイト数は、ワクチン群では平均２０４万匹／ｍｌで、対照群の平均１１０万匹／ｍｌより多かったが、むしろ差はないとみるのが適切と思われる。ヘマトクリット値（試験終了時）は、ワクチン群では平均２０．６であったのに対し、対照群では１６．３で、ワクチン群におけるヘマトクリット値の回復が対照群より早い傾向が見られた。更に、増体重はワクチン群では平均６０ｇであったのに対し、対照群では−４５ｇであった。
以上の試験結果から、特にＳＳＡ価の測定及び臨床症状の観察の結果から効果が認められると判定された。
【００６９】
試験−２
【００７０】
【表５】

表５の結果から明らかなように、ＥＬＩＳＡ抗体価は、ワクチン群では１００〜１２８００倍で、対照群の全例＜１００倍に比較して上昇が認められた。ＳＳＡ価は、ワクチン群では７例中３例に４〜３２倍の抗原が認められたが、残る４例には抗原は認められなかった。これに対し対照群では全例に３２〜２５６倍の抗原が認められた。
【００７１】
臨床症状観察結果では、ワクチン群では全例に死亡する例は認められなかったが、発症は全例に認められ、その程度はワクチン群では７例中１例が＋＋＋の重度の発症を示した他は１例が＋＋の中等度であり、５例は＋の軽度であった。これに対し対照群では７例中１例が＋＋の中等度以外は、残り６例が＋＋＋の重度であった（肉冠褪色、元気なくうずくまり、嗜眠状態の何れか１つの症状を示した場合は＋、２つの症状を示した場合は＋＋、全ての症状を示した場合は＋＋＋とした）。
【００７２】
ガメトサイト数は、ワクチン群では１６３万匹／ｍｌで、対照群の６０１万匹／ｍｌに比較して少なかった。赤血球数は、ワクチン群では３１１万個／ｍｍ^３ であったのに対し、対照群では２２４万個／ｍｍ^３ であった。更に増体重は、ワクチン群では平均１４０ｇであり、対照群の平均４３ｇを上回った。
以上の結果から、特にＳＳＡ価の測定および臨床症状の観察結果から効果が認められると判定された。
【００７３】
試験−３
【００７４】
【表６】

表６の結果から明らかなように、ＥＬＩＳＡ抗体価は、ワクチン群では１００〜８００倍で、対照群の全例＜１００倍に比較して上昇が認められた。ＳＳＡ価は、ワクチン群と対照群と共に臨床的に特に所見は認められなかった。臨床症状観察結果では、ワクチン群と対照群と共に、臨床的に特に所見は認められなかった（表中の＋は、肉冠に軽度の白色化がみられたことを示す）。
【００７５】
ガメトサイト数は、ワクチン群では全例ガメトサイトは認められず、これに対し対照群では平均６１２万匹／ｍｌで、両者には明らかな差が認められた。増体重は、両者間に差は認められなかった。なお、この試験では、ワクチンの１回に１ｍｌ接種でも３ｍｌ接種でも効果に差は認められなかった。
以上３例の試験成績より、本ワクチンはロイコチトゾーン症ワクチンとして効果が認められると判定された。
【００７６】
【配列表】
【００７７】
配列番号：１
配列の長さ：２０１９
配列の型：核酸
トポロジー：直鎖状
配列の種類：塩基配列
起源
生物名：ロイコチトゾーン・カウレリー第２代シゾント
株名：静岡株
配列
【００７８】
【化２１】

【化２２】

【化２３】

【００７９】
配列番号：２
配列の長さ：３３９９
配列の型：核酸
トポロジー：直鎖状
配列の種類：塩基配列
起源
生物名：ロイコチトゾーン・カウレリー第２代シゾント及び大腸菌プラスミドｐＢＲ３２２由来発現プラスミドｐＫＫ２３３−２由来ｐＭＡＬ−ｃＲＩ
株名：静岡株
配列
【００８０】
【化２４】

【化２５】

【化２６】

【化２７】

【図面の簡単な説明】
【図１】ロイコチトゾーン・カウレリー第２代シゾントの免疫原性蛋白質遺伝子のクローニング及びワクチンクローンの選定の工程図である。
【図２】ロイコチトゾーン・カウレリー第２代シゾント免疫原性蛋白質遺伝子のクローニングを示すものである。
【図３】クローン化第２代シゾント免疫原生蛋白質遺伝子の発現ベクターｐＭＡＬ−ｃＲＩへの挿入を示すものである。
【図４】Ｒ７及びＲ１５及びＲ４ａプローブをそれぞれに対するｐＵＣ１９−クローンプラスミド反応のハイブリダイゼーションを示すものである。
【図５】ｐＭＡＬ−ｃＲＩ−Ｒ７切断Ｒ７フラグメントのアガロース電気泳動像を示すものである。
【図６】（ａ）および（ｂ）はＴＢ１／ｐＭＡＬ−ｃＲＩ−Ｒ７発現材料のＳＤＳ−ＰＡＧＥ像を顕微鏡写真によって示すものである。
【図７】ＴＢＩ／ｐＭＡＬ−ｃＲＩ−Ｒ７発現材料のウエスタンブロット像を顕微鏡写真によって示すものである。
【図８】発現材料のカラムクロマト画分のＳＤＳ−ＰＡＧＥ像を顕微鏡写真によって示すものである。
【図９】発現材料のカラムクロマト画分のウエスタンブロット像を顕微鏡写真によって示すものである。[0001]
[Industrial applications]
The present invention relates to a vaccine produced by a genetic engineering technique against chicken leukocytosonosis. More specifically, the present invention relates to a leucocytozoidae, a leukocytozoidae family that belongs to the family Leucocytozoidae, which belongs to the genus Leucocytozoonsis, which is classified into the order Eucoccidae and schistosporids (subfamily). Immunogen sex The present invention relates to a vaccine against chicken leukocytosonosis using, as an active ingredient, a protein expressed by a host cell into which an expression vector into which a recombinant DNA molecule encoding a protein has been inserted.
[0002]
[Prior art]
Leukocyte cytosonosis in chickens is a disease caused by infection of the schistosporid Leucocytozon cauleryi. L. cauleryi was first recognized in Vietnam in 1909 as a round gametocyte (reproductive mother) without malaria granules in chicken blood, and was so named as a protozoan of the genus Leucocytozone.
[0003]
Leukocytosonosis in chickens in Japan was first confirmed in Hyogo Prefecture in 1954, and its pathogen was L. cerevisiae. cauleryi, and thereafter, the protozoan is experimentally found to be biologically transmitted by the chicken deer Culicoides arakawae (hereinafter abbreviated as "Nukaka") belonging to the diptera species Ceratopogonidae, a kind of blood-sucking insect. It was proved, and the life cycle was almost revealed. Leucocytozone cowlery has three developmental phases: dizogony (increased reproduction) and gametogony (reproductive and reproductive) in the host chicken, and sporogoni (spore-reproduced) in the body of the transmitter, Nukaka. ing.
[0004]
Sporozoites (species insects) that have entered the chicken body together with their saliva when sucking blood from the bran infected with the Hongen, parasitize cells of the vascular endothelial system such as lung, liver, spleen, and kidney as the first species. Form a fission schizont (split). From day 5 to day 7 after infection, the first generation merozoites (split bodies) are released into the blood, which re-enter the vascular endothelial cells distributed throughout the body and proliferate as second generation schizonts, As described below, it further develops in the intercellular space away from the host cell. On day 14, these schizonts release second generation merozoites into the blood, which then invade red blood cells and develop, and on days 18-19, are released from host cells and release macrogametocytes (female, respectively). Reproductive mother), matures as microgametocytes (male reproductive mother).
[0005]
Both gametocytes are taken into the midgut together with chicken blood when sucking Nukaka, form macrogamates (female reproductive bodies) and microgamates (male reproductive bodies), fuse (fertilize), and oginate through tigote (fused body) (Intestinal lobe), penetrates into the intercellular space of the worm intestinal wall, and then migrates below its outer membrane to develop into oocysts (cysts). Dozens of sporozoites are formed inside the oocyst, Collapse Then, it moves to the salivary gland after coming out of the contralateral space of Nukaka to complete its growth cycle.
[0006]
Symptoms and lesions in chickens caused by this protozoan infection are mainly observed at the end of schizogony, in which the second generation schizont develops, and at the time of gammetony, in which the second generation merozoites develop into gametes by parasitizing erythroid cells. It is thought that the most characteristic bleeding lesions and anemia in each organ and tissue of this disease are caused by bleeding associated with vascular embolism by the second generation schizont and the destruction of erythrocytes due to parasitism of the gametogony parasite. La Have been.
[0007]
As the vector of the protozoan, Nukaka lives in paddy fields and so on, and it is recognized as endemic in Southeast Asia, China, North Korea and South Korea which have paddy fields. In Japan, the first epidemic of leukocytosonosis has been confirmed, and the epidemic has been repeated every summer, causing a large loss in chicken productivity. Since 1964, pyrimethamine has been used since 1968. Since the combination of pyrimethamine and a sulfa drug was added to the sample as a preventive agent for this disease, the incidence has dramatically decreased. However, the use of active drugs was limited due to the protozoan's resistance to these drugs and the persistence of the drugs, and the use of some active drugs was banned due to safety issues. It has become difficult to withstand such damages, and the damage has become permanent.
[0008]
Conventional vaccines against chicken leukocytosonosis include live vaccines using sporozoites, inactivated vaccines using substances derived from insect bodies as antigens, and inactivation using organ emulsions from chickens infected with protozoa. There is a vaccine. A live vaccine using sporozoites isolates sporozoites from salivary glands of brassicae and immunizes chickens with a small number of them (Shibara et al., Shizuoka Prefectural Poultry Laboratory Research Report, 13: 25-27 (1978)). Inactivated vaccines using insect body-derived substances as antigens are as follows.
[0009]
Insect-derived antigens include first-generation schizonts, first-generation merozoites, second-generation schizonts, second-generation merozoites, and soluble antigens derived from chicken bodies and embryonated hen eggs, of which 10 to 15 days after inoculation with sporozoites Soluble antigens detected in the sera of infected chickens have almost the same antigenic properties as the second generation schizont, and likewise a relatively strong immunogen. sex Has been recognized. Thus, a vaccine produced by inactivating the serum antigen material with formalin has been recognized for its usefulness [Isobe and Suzuki, Jpn, J. Am. Parasitol. , 37: 214-219 (1988)].
[0010]
Further, the organ emulsion inactivated vaccine was made to improve the difficulty in obtaining a large amount of the protozoan antigen as described above, and the thymus, lung, liver, spleen, kidney, and kidney were obtained from infected chickens 13 days after inoculation with sporozoites. F sac is collected and used as an organ emulsion, and a vaccine is produced on a trial basis using a material inactivated with formalin, and its effect has also been recognized [Morii, T. et al. et al. , J. et al. Parasitol. Res. , 76: 630-632 (1990)].
[0011]
[Problems to be solved by the invention]
As described above, although the effectiveness of the vaccine has been confirmed for the time being, it is difficult to mass-produce as described above, and there is a problem with supply. As a result, the current situation still requires the reliance on synthetic drugs, and thus the above-mentioned problems have arisen. To cope with this, new drugs need to be developed, but vaccines that do not cause persistence problems because they are used in laying hens are desired. But the protozoa Bacteria Vaccines that are effective and highly safe because of their complicated life cycle and difficult to culture compared to viruses and viruses, and that can be supplied at low cost in mass production of Currently, no development has been done yet.
[0012]
However, since the life cycle of the protozoan was clarified, artificial breeding technology for brassicae was established, sporozoites could be secured, and protozoal infection experiments on chickens became possible at the same time. In addition, it became possible to collect the protozoa at each stage of the protozoan development cycle in the chicken. These results have made it possible to carry out immunological studies on leukocytosonosis, measure antibody titers in blood of infected chickens, and have confirmed the appearance of specific antigens in serum and the production of antibodies against them. In addition, results have been obtained that suggest the existence of immunogenicity in the second generation schizonts of protozoa. Therefore, in order to minimize the loss in the poultry industry with a preventive method against this disease based on such basic test research, there is a strong need for stable and mass-produced supply of effective and inexpensive vaccines.
[0013]
To prevent chicken leukocytosonosis, the above-mentioned prophylaxis has considerable limitations in general practical application. In other words, immunization with sporozoites produces sporozoites from brassicae, so there are difficulties in supplying them in large quantities and preserving sporozoites, and since brassica is also known to be a vector insect for infectious diseases other than this disease, Other pathogens that may be lurking in the wild can also be transferred to the chicken. In addition, chickens that have been experimentally infected must always be used as a material to obtain serum antigens and organ emulsions, and vaccines based on these have low production efficiencies.
[0014]
In view of the above, an object of the present invention is to prepare a vaccine using a recombinantly expressed protein produced by a genetic engineering technique in order to prevent leukocytosonosis without using chicken and nuka. An object of the present invention is to provide a gene clone expressing a chicken leukocytozone immunogenic protein, which can be supplied stably in large quantities at a low cost, and a recombinant vaccine against chicken leukocytozone disease.
[0015]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides a method for isolating and purifying a second generation schizont (2GS) formed in schizogonies in the developing stage of Leucocytozone kaurely, which is believed to contain an antigen important for infection protection, From this, mRNA was extracted and purified, cDNA for this was prepared, and then a cDNA library was prepared by in vitro packaging using lambda phage λgt11. Next, Escherichia coli Y1090 strain was transformed with this cDNA library, and immunogenic protein gene clones were selected by immunoscreening using a monoclonal antibody against 2GS antigen.
[0016]
The clone-derived gene was inserted into the expression vector pMAL-cR1, and the protein (glutamic acid 45) of the immunogenic protein gene (R7 gene, R7: derived from the clone number) of the recombinant microorganism in which the inserted expression plasmid was transferred to the E. coli TB1 strain. (Acid protein containing 0.1%) was expressed as a fusion protein with maltose-binding protein, and an ultrasonic extract of cells containing this protein was formulated.
[0017]
The clones were selected by immunoscreening using a monoclonal antibody prepared using a 2GS ultrasonic extract as a starting material. This monoclonal antibody was previously immunized with 2GS antigen purified on a monoclonal antibody-binding affinity column. It was confirmed that it had the ability to give a gene, and it was confirmed that it was effective in selecting 2GS immunogenic protein genes. The protein expressed by the R7 gene reacts by ELISA with both the monoclonal antibody used for clone selection and the chicken serum infected with leukocytozone protozoa, and chickens immunized with the protein are resistant to leukocytozone infection. To gain resistance. The present invention has been completed based on such findings.
[0018]
In the present invention, the cDNA to be inserted into the vector is not particularly limited as long as it is derived from a protozoan classified as Leukocytozone caulerie. In the present invention, the immunogenic protein gene clone specifically refers to a protein library that recognizes an immunogenic protein from a cDNA library prepared from mRNA encoding a protein possessed by the second schizont of leukocytozone protozoa. A part of the clones obtained by the recognition by the monoclonal antibody described above, and a clone which reacts with a part of the clones by Southern hybridization, and has substantially the following nucleotide sequence.
[0019]
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[0020]
The vectors that can be used in the present invention include Escherichia coli expression vectors, Escherichia coli phage-based vectors, Escherichia coli-cultured cell shuttle vectors, and viral vectors, which are particularly limited as long as they can express proteins in host cells, particularly Escherichia coli. Not done. When a vaccine against leukocytosonosis is prepared using the expressed protein of the present invention, a protective effect is observed even when an infected cell extract is used as it is without any purification operation. The concentration is, for example, in the case of expression in Escherichia coli.However, the degree of the concentration depends on the amount of suspension in centrifugal precipitation of the culture solution during sonication or the amount of lysate of ammonium sulfate concentrated precipitate in the ultrasonic extract. it can. Chicken can be immunized by admixing, for example, an aluminum hydroxide gel as an adjuvant by a known method.
[0021]
The raw materials and preferred combinations used in the present invention are described below.
(1) Recombinant microorganism TB1 / pMAL-cR1-R7
This stock is stored at -20 ° C as a master cell stock and a working seed stock, and serves as a starting material for producing vaccines.
(2) Ampicillin
The expression vector pMAL-cR1-R7 transferred to the host Escherichia coli TB1 strain has an ampicillin resistance gene, which confers one selectivity on the culture of the host bacterium.
(3) L-broth agar medium
A medium for performing one-dimensional culture from working seeds, containing 1% tryptone, 0.5% yeast extract and 1% sodium chloride as main components, 1% bacterial agar powder and 100 μg / ml ampicillin.
[0022]
(4) L-broth medium, pH 7.0
A medium for performing two-dimensional culture from one-dimensional culture, which is obtained by removing agar powder from the above L-broth agar medium.
(5) L-broth expression culture medium, pH 7.0
A medium for expressing and culturing a recombinant microorganism. The L-broth medium further contains 0.2% glucose.
(6) isopropyl-β-D-thiogalactopyranoside
An expression inducer added to a culture solution during the logarithmic growth phase during expression culture.
(7) lysis buffer, pH 7.0
A buffer for suspending cells when sonicating cultured cells, the buffer containing 10 mM phosphate, 30 mM sodium chloride, 0.25% tween 20, 10 mM 2-mercaptoethanol, 10 mM EDTA, and 10 mM EGTA.
[0023]
(8) Saturated ammonium sulfate
The active ingredient of the ultrasonic extract of the cells is precipitated.
(9) Physiological buffered saline (PBS), pH 7.0
Dissolve the precipitate of active ingredient with saturated ammonium sulfate.
(10) Formalin
Add to a final concentration of 0.2% for inactivation and preservation of the vaccine.
(11) Aluminum hydroxide
As a vaccine adjuvant, add to a final concentration of 500 μg A1 / ml.
[0024]
General usage of raw materials used in the present invention, preferred range of usage
(1) Working seed stock of recombinant microorganism TB1 / pMAL-cR1-R7: loopful amount of Ezel or several μl at a time
(2) Ampicillin: 100 μg / ml
(3) L-broth agar medium, pH 7.0: 1 plate of 20 ml / plate at a time
The following are the amounts used for 100 l cultures of recombinant microorganisms and their treatment.
(4) L-broth medium, pH 7.0: 2 l
(5) L-broth expression culture medium, pH 7.0: 100 l
(6) Isopropyl-β-D-thiogalactopyranoside: 1 M solution, 100 ml
[0025]
(7) lysis buffer, pH 7.0: 6.7 l (about 1/15 of the expression culture)
(8) Saturated ammonium sulfate: 6.7 l (same amount as lysis buffer)
(9) PBS, pH 7.0: 5 l
(10) Formalin: 10%, 100 ml × 2 (including the amount added after the addition of the following aluminum hydroxide gel)
(11) Aluminum hydroxide gel: 1000 μg A1 / ml, 5 l
[0026]
[Action and effect]
(1) Action
Since the vaccine according to the present invention is a product of a transformed microorganism, it is superior to the protozoan antigen collection method produced in chickens in the following points.
1) Safety: Since the recombinant microorganism used in this vaccine is not pathogenic, toxic, or communicable, the vaccine does not have such properties. In addition, this vaccine is an extract obtained by sonication of a recombinant microorganism and is inactivated by formalin, so that there is no possibility of infectivity.
2) Economical: It is excellent in economical efficiency because it can omit all of nukaka and chicken for collecting protozoa and breeding facilities, facilities, and manpower required for them.
3) Productivity: Since the host bacterium of the recombinant microorganism is Escherichia coli, large-scale cultivation can be easily performed, and the operation of purifying the expressed protein is not required, so that the productivity is high.
[0027]
(2) Effect
1) After immunization, as described in Example 4, sporozoites were inoculated intravenously into 13,000 to 15,000 chickens per chicken, and the vaccine was experimentally infected. Because of the suppression, it meets this purpose.
2) There is no problem of pathogen contamination to the environment from the viewpoint of safety.
3) Since the ELISA antigen titer of each sample by the expression culture is almost constant, it is possible to provide a uniform vaccine with little quantitative variation of the antigen.
[0028]
【Example】
EXAMPLES Examples of the present invention will be specifically described below, but it goes without saying that the present invention is not limited by these examples.
Example
Cloning of the immunogenic protein gene of Leukocytozone caulerie second generation schizont, selection of vaccine clones, and prevention of infection with recombinant vaccine
Functional test
(1) cDNA library derived from the second generation schizont of Leucocytozone and Cowlery
Preparation of
Sporozoites of Leucocytozone Kaurelie prepared from Nukaka were inoculated intravenously into about 10,000 chickens per chicken about 5 weeks old for experimental infection. On the 13th day of the appearance of the second schizont (2GS) after infection, chickens are dissected, thymus, lung, liver, kidney, spleen, and F sac organs are collected, and 2GS is separated from those organ emulsions And purified.
[0029]
From this, RNA was extracted by the guanidium isothiocyanate method, fractionated precipitation with lithium chloride was performed, and ethanol was further precipitated to obtain total RNA. Then, mRNA was extracted using Oligotex-dT30 (Takara Shuzo). Obtained by repeating purification twice. Next, cDNA was synthesized using a commercially available kit (manufactured by Amersham) using mRNA as a template, oligo (dT) nucleotides or random hexanucleotides as primers, and using reverse transcriptase according to the method of Gubler and Hoffman.
[0030]
The resulting single-stranded cDNA was reacted with RNase H and DNA polymerase to form a double-stranded cDNA, to which an EcoRI adapter (Takara Shuzo) was added, and ligated to both arms of lambda phage λgt11 (Amersham). Then, lambda phage was packaged using an in vitro packaging kit (manufactured by Stratagene) to prepare a cDNA library.
In preparing the cDNA library, the EcoRI adapter-added cDNA was size-fractionated into four by agarose electrophoresis. After ligation operation on these, in vitro packaging was performed. Accordingly, four cDNA libraries were prepared for each of the two primer cDNAs.
[0031]
(2) Plaque immunoscreening of cDNA library and selection of clones
Based on two primers each In the cDNA library, the smaller two species (<500 bp) of each size derived from the size fraction before the preparation were omitted, and the larger two species (> 500 bp) were each infected with E. coli Y1090 strain, Plaques were formed on an L-broth agar medium containing / ml of ampicillin in a conventional manner. According to a conventional method, the DNA was transferred to a nitrocellulose filter impregnated with isopropyl-β-D-thiogalactopyranoside (IPTG), which is an inducer for the lac promoter, and subjected to immunoscreening. As the primary antibody used for screening, an equal mixture of two monoclonal antibodies, M5 recognizing around 67 kDa of the 2GS antigen and R1 recognizing 100 to 200 kDa, was used. HRP-labeled rabbit anti-mouse IgG + IgM + IgA was used as the secondary antibody, and diaminobenzidine was used as the substrate for the color reaction.
[0032]
The monoclonal antibody is a test using a mixture of each of the 2GS antigens which has been previously purified on each monoclonal antibody binding affinity column. sex Was recognized and judged to be suitable for this screening. In order to prevent oversight of the screening, a mixture of the two was used.
In both cases, the antigens recognized by them are not single in molecular weight, but each of the above-mentioned certain ranges is recognized, so that the protozoan antigen has a repeating structure of the same epitope. It is inferred.
[0033]
As a result of cloning, 33 types (labeled with T numbers) of cDNA based on oligo (dT) nucleotide primers and 44 types (labeled with R numbers) of cDNA based on random hexanucleotide primers was gotten. After re-cleaning them, clones were selected.
[0034]
That is, after re-screening, the phage that showed a relatively strong positive reaction in color development were proliferated, and then infected with Escherichia coli strain Y1089 to prepare a lysogen, followed by expression culture using IPTG as an inducing agent, A cell lysate was prepared and an immunodot blot test was performed using the same monoclonal antibody mixture to reconfirm the expression of the antigen.On the other hand, positive phages were grown and purified, and then confirmation of the production of EcoRI-cleaved fragments was performed. As a result, 17 kinds of R1a, R1b, R4a, R4b, R6, R7, R11, R13, R15, R18, R29, R35, R42, T3, T4, T9, and T10 were selected as potential clones of the immunogenic protein gene. did. A summary of the above steps is shown in FIGS. 1, 2 and 3.
[0035]
In order to determine whether the clones thus obtained can actually impart immunogenicity, at this stage, 500 μg of aluminum hydroxide gel was used as an adjuvant for any two cell lysates of R15 and T10. / Ml to test the infection protection test. The outline of the method of the infection protection test and the method of judging the effect are described in Example (4), but the results of the infection protection test of the expressed proteins of the R15 and T10λgt11 clones include the uninoculated control group as shown in Table 1. No onset or death was observed in any of the cases. However, after the immunized chickens were challenged with 19,500 sporozoites / wing, serum soluble antigen (SSA) derived from the developmental process of protozoa appearing in the serum on day 13 was observed. With regard to the quantitative value (SSA value) of (1), the number of birds having a low value was higher in the immunized group than in the non-inoculated control group, and thus it was determined that R15 and T10 had weak but immunity.
[0036]
It was also found that the vaccine antibody titer (ELISA antibody titer) against the 2GS antigen in the serum immediately before the challenge was higher in the immunized group than in the uninoculated control group in all cases. From this, the clone obtained by this screening was judged to be suitable for this purpose.
[0037]
[Table 1]

[0038]
(3) Selection of expression vector pMAL-cRI subclone and vaccine clone For expression culture for practical use of vaccines, the genes of the above 17 clones were inserted into expression vector pMAL-cRI (manufactured by Biolabs), respectively. A pMAL-cRI subclone was obtained by the same immunoscreening as described above, and was transferred to E. coli TB1 strain. Vaccine clones were selected from these subclones by categorizing the clones by ELISA titration against each of the two monoclonal antibodies and Southern hybridization. A sample prepared by performing expression culture was used for ELISA, and a plasmid prepared by subcloning the gene into pUC19 was used for Southern hybridization.
[0039]
The method for preparing a sample by expression culture, which is also a method for preparing a preparation, is as follows. In a logarithmic growth phase, IPTG was added to a final concentration of 1 mM in shaking culture at 37 ° C. using L-broth liquid medium of the host bacterium, followed by further culturing at 37 ° C. for 4 to 6 hours to convert the protein to maltose-binding protein. Was expressed as a fusion protein, and the cells obtained by centrifugation were suspended in a lysis buffer, subjected to ultrasonic treatment, and centrifuged again to obtain an ultrasonic extract. Further, the precipitate obtained by treating this extract with saturated ammonium sulfate was dissolved in PBS, formalin was added to a final concentration of 0.2%, and the mixture was allowed to stand at 4 ° C. for at least one night to prepare a sample.
[0040]
As a result of the measurement of the ELISA antigen titer, 8 out of 17 clones showed clear reactivity with either of the monoclonal antibodies M5 and R1, and among them, the monoclonal antibody M5 reaction groups were R1, R4a, and R13. At T3 and T3, the monoclonal antibody R1 reaction groups were R7, R15, R35, and T10, all of which were four types. In addition, R7 and R15 which showed strong reactivity with the monoclonal antibody R1 also showed reactivity with the monoclonal antibody M5 depending on the culture.
[0041]
ELISA antibody titer of expressed protein (crude substance) of pMAL-cRI subclone and group size and clone size by Southern hybridization, and ELISA antigen titer: reactivity to monoclonal antibodies M5 and R1, respectively; Southern hybridization: probe R4a , R7 and R15 are shown in Table 2.
[0042]
On the other hand, as a result of Southern hybridization using the purified fragments derived from the plasmids R4a, R7 and R15 as probes, as shown in FIG. 3, R4a and T3 for the probe R4, and any of the probes R7 and R15 for the probe R4. In each case, R7, R15, R35 and T10 showed reactivity. These are shown in Table 2 together with the DNA size (approximate value) of each clone.
[0043]
[Table 2]

[0044]
In the Southern hybridization, those that reacted with the probe R4a were R4a and T3, and no reactivity with R1a and R13, which was the same group as the monoclonal antibody M5 reaction group by ELISA titer, was observed. Was determined to be consistent with the grouping of clones recognizing the two monoclonal antibodies according to the above and the grouping of clones showing mutual similarity of genes by Southern hybridization.
[0045]
Thus, an attempt was made to compare infection protection tests using a mixture of expression samples of each of the four clones in the monoclonal antibody R1 recognition group and the M5 recognition group. The results are shown in Table 3. In the ELISA antibody titer against 2GS antigen of the serum at the time of sporozoite challenge, the R1 group clearly exceeded the M5 group, and the degree of suppression of SSA on the 13th day after challenge was significantly higher in the R1 group than in the PBS control group. Furthermore, from the results of infection protection performed by arbitrarily selecting eight individual clones, the immunizing antibody of the representative clone R4a reacting with the monoclonal antibody M5 reacts with the same R4a antigen by ELISA, but the 2GS antigen Did not always show reactivity, whereas the immunized antibody produced by the clone reacting with the monoclonal antibody R1 was found to show reactivity with the same antigen and 2GS antigen by the same method.
[0046]
On the other hand, from the results of the eight types of Western blots, the monoclonal antibody M5 also recognizes many proteins in an extract (negative expression control) derived from a host cell into which a vector into which the gene was not inserted was transferred. In contrast, the monoclonal antibody R1 did not show such a phenomenon, and was found to always recognize mainly the protein produced by the expression.
[0047]
[Table 3]

[0048]
From the above, it was determined that it was appropriate to select a vaccine clone from an R1 monoclonal antibody-recognizing clone showing specificity for the 2GS antigen. As a result of further examination of these clones in the infection protection test, both R7 and R15 clones were listed as vaccine clone candidates, but R7, which exhibited more effects than other clones, was finally determined as a vaccine clone. The recombinant microorganism of the R7 clone obtained in this manner was designated as TB1 / pMAL-cR1-R7, and a vaccine produced by expressing the recombinant microorganism was called a leukocytosonosis gene recombinant vaccine (abbreviated as R7 vaccine). The recombinant microorganism TB1 / pMAL-cR1-R7 has been deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology on March 22, 1994 under the accession number FERM P-14240.
[0049]
Next, the following results will be described for the present microorganism TB1 / pMAL-cR1-R7.
Electrophoresis image of pMAL-cR1-R7 cleaved R7 fragment:
Proliferation of the recombinant microorganism TB1 / pMAL-cR1-R7 The pMAL-cR1-R7 plasmid purified from the culture and the EcoRI-cut DNA thereof were subjected to 1.2% agarose gel electrophoresis and stained with ethidium bromide. FIG. 4 shows that the pMAL-cR1-R7 plasmid was obtained from the recombinant microorganism, and an EcoRI-cleaved plasmid produced a vector of about 6.1 kbp and an R7 fragment of about 2.0 kbp.
[0050]
Electrophoresis image and waste of expression material of this microorganism TB1 / pMAL-cR1-R7
Tan blot image:
A 1/10 volume of the sonicated extract of the cell suspension was further treated with saturated ammonium sulfate to a 1/40 volume of the expression medium, and an equal volume of the sample buffer for electrophoresis was added to the expression material. , And 20 μl of the mixture was applied to an SDS-polyacrylamide gel (8% concentration gel) to perform electrophoresis (SDS-PAGE). The monoclonal antibody R1 was used as the primary antibody in the Western blot. The SDS-PAGE image at this time is shown in FIGS. 6A and 6B, and the Western blot image is shown in FIG.
[0051]
As shown in FIGS. 6 (a) and 6 (b), two bands appeared near 200 kDa in the expressed material, whereas those bands were not observed in the non-expressed material in which expression was not induced. In addition, as shown in FIG. 7, the expression material showed reactivity in those bands, while the non-expression material showed no reactivity. The reactivity of the expression material at a 20-fold dilution was considered to be only one of the two bands.
[0052]
As described above, a material having a band near 200 kDa shows an ELISA titer by a reaction with the monoclonal antibody R1 described below. In order to further confirm the relationship between them, the expression material was subjected to column chromatography using Sephacryl S200. The SDS-PAGE image and the Western blot image were similarly examined for the 4-fold concentrated material with respect to the column applied amounts of the fractions F1, F2 and F3. The SDS-PAGE image and the Western blot image at this time are shown in FIGS. 8 and 9, respectively.
[0053]
In SDS-PAGE, two bands near 200 kDa in F1 also showed Western blot reactivity, but no reactivity was observed in F2 and F3 where these bands were not observed. In addition, the reactivity at 20-fold dilution of F1 seems to be only one of the two bands. In addition, ELISA titers of these fractions were observed in F1, but not in F2.
[0054]
The ELISA titers of the expression material and its filtered fraction (ELISA titers using monoclonal antibody R1 diluted 100-fold) are as follows.

[0055]
Thus, in the expressed material, bands appeared near 200 kDa in SDS-PAGE, and they showed a Western blot reaction, whereas in the same non-expressed material, those bands did not appear, and the reactivity was also observed in the Western blot. Since these were not observed and the ELISA antigen titer was observed only in the material in which these bands appeared, those substances were determined to be expressed fusion proteins. The base sequence of the R7 gene is as follows, and the underlined portions at both ends indicate EcoRI adapter regions.
[0056]
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[0057]
Expression fusion protein region of the present microorganism TB1 / pMAL-cR1-R7
The fusion protein region
[0058]
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As shown in the above, it is 3399 bp (1132 amino acids) from the first start codon ATG of the maltose binding protein gene to the last stop codon TGA of the 1acZa gene. The 2019 bp R7 gene is inserted therein via an EcoRI adapter. The EcoRI adapter region is indicated by a broken line, and the R7 region is indicated by a solid line. The protein in the region including the EcoRI adapter at both ends of the R7 gene is composed of 689 amino acids and has the following composition.
[0059]
Ala 3 (0.4) Leu 2 (0.3)
Arg 2 (0.3) Lys 68 (9.9)
Asn 25 (3.6) Met 5 (0.7)
Asp 12 (1.7) Phe 2 (0.3)
Cys 1 (0.1) Pro 5 (0.7)
Gln 14 (2.0) Ser 6 (0.9)
Glu 311 (45.1) Thr 54 (7.8)
Gly 9 (1.3) Trp 0 (0.0)
His 68 (9.9 Tyr 8 (1.2)
Ile 35 (5.1) Val 59 (8.6)
End 0 (0.0)
[0060]
Acidic amino acid (Asp + Glu) 323 (46.9)
Basic amino acid (Arg + Lys) 70 (10.2)
Aromatic amino acid (Phe + Trp + Tyr) 10 (1.5)
Hydrophobic amino acid (aromatic amino acid + Ile + Leu + Met + Val) 111 (16.1)
Molecular weight = 84144
[0061]
(4) Infection protection test of this R7 vaccine
To determine the efficacy of the protozoan vaccine by the infection protection test, the immunized chickens were challenged with sporozoites, and then the serum soluble antigen (SSA) titer was measured mainly on the 13th day, and the onset, life / death determination up to the 21st day was performed. The results of evaluation, such as observation of clinical symptoms, including the measurement of the number of gametosites in blood on day 21 and the like, were evaluated. Other measurements of vaccine antibody titers (serum antibody titers immediately before the challenge) and 21 days after the challenge The results of the measurement of the number of red blood cells in blood were also used as a reference for evaluation.
[0062]
The measurement of the SSA value or the number of gametosites is to be evaluated even when sporozoites develop on the 21st day after the attack, and when the effect cannot be determined by life or death. The SSA titer was determined by examining the amount of SSA titer derived from the protozoan development process appearing in the serum by the attacking sporozoites by the sedimentation reaction in a gel using infected chicken-resistant serum. In other words, the growth of sporozoites was suppressed, and thus the protective effect was recognized as the number of birds with a small response increased as compared with the number of birds in the non-immune control group. Similarly, the measurement of the number of gametocytes also measures the number of sporozoites that develop into gametocytes, To The smaller the number of birds is, the larger the number of birds is compared to the number of non-immune control groups, the more the protective effect was recognized.
[0063]
The vaccine was prepared as described in (3) of the above-mentioned Example, and when the aluminum hydroxide gel was finally added, the amount was reduced to 1/10 of the amount of the culture solution. Hereafter, the three prototypes were tested as Test-1, Test-2 and Test-3, respectively. In Test-1, Test-2 and Test-3, SPF chicken-derived chicks were used for all 36-, 23-, and 21-day-old chickens, and 0.5 ml of the vaccine was measured in the left and right thigh muscles. 1 ml was injected twice at two week intervals. In Test-3, further attempts were made with a total of 3 ml of 1.5 ml each. Two weeks after the second injection of the vaccine, serum was collected, and the challenge was intravenous inoculation of 13600 sporozoites of Shizuoka strain of Leuco parasite, 12,500 in Test-1, 12,500 in Test-2, and 15,000 in Test-3. I went.
[0064]
Blood was collected immediately before the challenge, clinical symptoms were observed for 21 days after the challenge, blood was collected on the 13th day, and a blood sample was collected at the end of the observation. Furthermore, the body weight was measured at the time of the challenge and at the end of the test.
The vaccine antibody titer (serum antibody titer by ELISA immediately before the challenge), the SSA value on the 13th day after the challenge, the observation of clinical symptoms up to the 21st day after the challenge in Test-1, Test-2 and Test-3 by them. Tables 4, 5 and 6 show the results of the number of gametosites and the weight gain at the end of the test, respectively.
[0065]
Test-1
[0066]
[Table 4]

1) Highest dilution of positive serum
2) Highest dilution of serum with sedimentation line
3) Days after attack
4) Inability to test for death
5) Unable to test due to blood coagulation
[0067]
As is clear from the results in Table 4, the ELISA antibody titer was <100- 1600 In all cases, an increase was observed as compared to <100 times in all cases in the control group. Regarding the SSA value, the antigens observed in the vaccine group were 2 to 32 times (average 14.5 times), while those in the control group were 8 to> 512 times (average 64.0 times). , Obvious between the two What Differences were noted.
[0068]
According to the clinical symptom observation results, 3 out of 8 cases occurred in the vaccine group and 2 of them died, whereas all of the 8 cases developed in the control group and 4 of them died. The number of gametosites averaged 2.04 million / ml in the vaccine group and more than the average 1.1 million / ml in the control group, but it seems appropriate to see that there is no difference. The hematocrit value (at the end of the test) averaged 20.6 in the vaccine group, while it was 16.3 in the control group. The hematocrit value in the vaccine group tended to recover more rapidly than in the control group. Furthermore, the weight gain was -45 g in the control group compared to an average of 60 g in the vaccine group.
From the above test results, it was determined that the effect was particularly recognized from the results of the measurement of the SSA value and the observation of the clinical symptoms.
[0069]
Test-2
[0070]
[Table 5]

As is clear from the results in Table 5, the ELISA antibody titer was 100 to 12800-fold in the vaccine group, and was higher than that in all cases in the control group <100-fold. Regarding the SSA value, in the vaccine group, the antigen was observed 4- to 32-fold in 3 out of 7 cases, but the antigen was not observed in the remaining 4 cases. On the other hand, in the control group, 32- to 256-fold antigen was observed in all cases.
[0071]
According to the clinical symptom observation results, none of the cases died in the vaccine group, but all cases showed onset, and the degree of occurrence was +++ in 7 cases in the vaccine group. Others were moderate in ++ in 1 case and mild in + in 5 cases. In contrast, in the control group, 1 out of 7 cases was ++ except for moderate ++, and the remaining 6 cases were ++ severe. Is +, when two symptoms are shown, and when all the symptoms are shown, +++).
[0072]
The number of gametocytes was 1.63 million / ml in the vaccine group, which was smaller than that of the control group (6.101 / ml). The red blood cell count was 31.11 million / mm in the vaccine group. ³ Whereas the control group had 2.24 million pieces / mm. ³ Met. Furthermore, the body weight gain was 140 g on average in the vaccine group, and exceeded 43 g on average in the control group.
From the above results, it was determined that the effect was particularly recognized from the measurement of the SSA value and the observation of the clinical symptoms.
[0073]
Test-3
[0074]
[Table 6]

As is clear from the results in Table 6, the ELISA antibody titer was 100-800-fold in the vaccine group, and was higher than that in all cases in the control group <100-fold. No clinically specific SSA value was observed in the vaccine group and the control group. In the clinical symptom observation results, no particular clinical findings were observed in the vaccine group and the control group (+ in the table indicates that mild whitening was observed in the crown).
[0075]
In the vaccine group, the number of gametocytes was found in all cases. Not Whereas the control group averaged 6.12 million Animal / ml Thus, a clear difference was recognized between the two. There was no difference in weight gain between the two. In addition, this of In the test, 1 ml of vaccine was inoculated at a time. 3 ml inoculation No difference in effect was observed.
From the test results of the above three cases, it was determined that this vaccine was effective as a leukocytosonosis vaccine.
[0076]
[Sequence list]
[0077]
SEQ ID NO: 1
Sequence length: 2019
Sequence type: nucleic acid
Topology: linear
Sequence type: base sequence
origin
Organism name: Leukotito Zone Cowlery 2nd schizont
Stock name: Shizuoka stock
Array
[0078]
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[0079]
SEQ ID NO: 2
Sequence length: 3399
Sequence type: nucleic acid
Topology: linear
Sequence type: base sequence
origin
Organism name: pMAL-cRI derived from expression plasmid pKK233-2 derived from Escherichia coli plasmid pBR322 and the second generation schizont of Leukocytozone caulerie
Stock name: Shizuoka stock
Array
[0080]
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[Brief description of the drawings]
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a process diagram of cloning of an immunogenic protein gene of Leukocytozone caulelli second generation schizont and selection of a vaccine clone.
FIG. 2 shows the cloning of the gene for the second generation of schizont immunogenic protein of Leucocytozone caulerie.
FIG. 3 shows the insertion of the cloned secondary schizont immunogenic protein gene into the expression vector pMAL-cRI.
FIG. 4 shows the hybridization of the pUC19-clone plasmid reaction to R7 and R15 and R4a probes, respectively.
FIG. 5 shows an agarose electrophoresis image of a pMAL-cRI-R7-cleaved R7 fragment.
6 (a) and (b) show SDS-PAGE images of TB1 / pMAL-cRI-R7 expressing material. By micrograph It is shown.
FIG. 7 is a western blot image of a TBI / pMAL-cRI-R7 expression material. By micrograph It is shown.
FIG. 8 shows an SDS-PAGE image of a column chromatography fraction of an expression material. By micrograph It is shown.
FIG. 9 shows a Western blot image of a column chromatography fraction of an expression material. By micrograph It is shown.

Claims (7)

The use of monoclonal antibodies that recognize an immunogenic protein derived from chicken Leucocytozoon, the immunogenic protein gene clones shown in SEQ ID NO: 1, characterized in that it is selected by immunoscreening. Immunogenic protein gene clones according to claim 1 is expressed, and a vaccine against chicken Leucocytozoon diseases, characterized in that it comprises an adjuvant. Recombinant DNA molecules you comprising a gene clone of claim 1 wherein. 4. The recombinant DNA molecule of claim 3, operatively linked to an expression control sequence. A vector into which the recombinant DNA molecule according to claim 4 has been inserted. The polypeptide of SEQ ID NO: 2 expressed by the recombinant DNA molecule of SEQ ID NO: 1 . A chicken leukocytosonosis vaccine prepared with the polypeptide of claim 6.

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