JP4953256B2 - 鳥類免疫系によって認識されるhn蛋白質のエピトープ及び前記エピトープ突然変異を含むニューカッスル病ウイルス - Google Patents
鳥類免疫系によって認識されるhn蛋白質のエピトープ及び前記エピトープ突然変異を含むニューカッスル病ウイルス Download PDFInfo
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- C07K2317/00—Immunoglobulins specific features
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Description
Morrison, T.,and A.Portner, Structure, function, and intracellular processing of the glycoproteins of Paramyxoviridae, 1991, p.347−375. In D.Kingsbyry(ed.), The Paramyxoviruses. Plenum Press, New York.
<1−1>ウイルス菌株の分離
ニューカッスル病ウイルス菌株は鶏から標準的方法(Alexander、1989)によって分離した。具体的には、気管(trachea)、前胃(proventriculus)、小腸(small intestine)及び盲腸扁桃(cecal tonsil)のような出血性病変部位のサンプルを使用してウイルスを分離した。前記分離したウイルスを10%のBCS(bovine calf serum、Gibco/BRL、Grand Island、NY)を添加したDMEM(Gibco/BRL、Grand Island、NY)培地で培養した鶏胚繊維芽細胞に接種し、細胞は5%のCO2、37℃の加湿環境下で培養した。
ウイルス同定及び線状抗原分析のためのHN部分塩基配列を、SEQ ID NO:10乃至15及び図5A乃至図5Fに示した。
ウイルスゲノムRNAは、酸−グアニジウム−フェノール(acid−guanidinium−phenol)法(Chomzinski and Sacci、1985)によって尿膜液から抽出した。具体的には、尿膜液100μlを1mlのTRI反応物(MRC co.,MA)に添加して抽出し、RNA沈殿物を0.1%のDEPC(ピロ炭酸ジエチル)処理された蒸留水(DW)50μlで溶かした。
HN蛋白質の遺伝子をABI377DNA自動分析装置とダイ・ターミネーター・キット(dye terminator kit、Perkin Elmer、Foster、CA)を使用して塩基配列を分析した(Kwon et al.,2000)。具体的には、PCR産物5μlを3M酢酸ナトリウム(pH5.2)1μl、95%のエタノール12.5μlに添加して氷に10分間静置した後、14,000rpmで15分間遠心分離し、70%のエタノールで洗浄した後、沈殿物を乾燥させてこれを1xPCRバッファー10μlに溶解させた。
前記で分析されたHN蛋白質の線状エピトープアミノ酸配列は、蛋白質−蛋白質BLAST検索(www.ncbi.nlm.nih.gov/blast)を通じて分析した。
<2−1>ペプチド合成
SEQ ID NO:1とSEQ ID NO:3に相当するペプチドをFmoc−化学法(Gloor et al.,1994)を用いて合成(ペップトロン社、大田、韓国)し、96穴プレートでコーティング効率を上げるために、SEQ ID NO:1のアミノ末端に7個のリシンを付け、SEQ ID NO:3のカルボキシル末端にはアミノカプロン酸(aminocaproic acid)4個を付けた。
ラソタウイルス(赤血球凝集力価1、024/25μl)は0.2%ホルマリンで不活化して、KBNP−4152(赤血球凝集力価248/25μl)は0.025%ホルマリンで不活化し、それぞれSPF発育卵5個に200μlずつ接種して3日間観察し、尿膜液を収穫して赤血球凝集検査を実施して陰性であることを確認した。フロイント完全アジュバントと不活化ウイルス同量を混合して油中に水を乳濁させた液を作ってそれぞれSPF鶏2匹に1匹当たり200μlずつ接種し、2匹は陰性対照群として実験期間の間つづけて飼育し、ニューカッスル病ウイルス抗体陰性の有無を確認した。ワクチン接種4週後、採血して血清を分離して冷蔵または冷凍して実験に使用した。
SEQ ID NO:2とSEQ ID NO:6で記載されるペプチドを50mMの炭酸塩・重炭酸塩(pH9.6)緩衝溶液に懸濁させて、96穴プレートの穴毎に125ngと4μgを室温で4時間コーティングした後、3%の牛血清アルブミン200μlを添加して4℃で一晩ブロッキングした。ブロッキング溶液をリン酸緩衝溶液(pH7.2)で洗浄した後、抗ラソタ血清と抗KBNP−4152血清を16倍、32倍、128倍、256倍希釈してそれぞれのペプチドがコーティングされた穴に添加して室温で1時間反応させた後、リン酸緩衝溶液で3回洗浄した。抗鶏免疫グロブリン−HRP結合2次抗体を添加した後、室温で1時間反応させてから、リン酸緩衝溶液で3回洗浄した後、基質溶液(TMB、3,3’、5,5’−テトラメチルベンジジン)を添加し、停止容液を入れて450nmの波長で吸光度を測定した。測定した吸光度は、血清を入れない穴の吸光度とペプチドをコーティングしない穴の吸光度を差し引きして数値を補正した。
ユニダイレクショナル(unidierctional)−赤血球凝集抑制検査のために、まずラソタ株とKBNP−4152の赤血球凝集力価を測定した。具体的には、96穴プレートのそれぞれの穴にリン酸緩衝溶液を50μlずつを噴霧し、第1穴にウイルス50μlを添加して混合した後、再び50μlを採取して次の穴に添加して混合する過程を繰り返し、最後の穴では50μlを採取して除去する方法で連鎖希釈を実施した。前記のようにウイルスが2倍単位で希釈された各穴に0.5%の鶏赤血球(v/v)50μlを添加して混合した後、4℃で30分間反応させて完ぺきな赤血球凝集が起こった穴の希釈倍数(2n、n=希釈倍数)を赤血球凝集力価にした。
ラソタ株とKBNP−4152接種で生成された抗体のKBNP−4152に対する中和能力を評価するために、ユニダイレクショナル(unidierctional)−ウイルス中和試験を実施した。つまり、試験管で血清をPBSで2倍希釈した後、96穴プレートに100μlずつ移して同量のウイルス(100 TCID50/25μl)を添加して37℃で1時間続けて中和させる。中和材料25μlと繊維芽細胞懸濁液100μlを各穴に接種する。37℃で4日間培養後に、完全中和が起こって細胞変性効果が全く現れない最高希釈倍数の逆数を中和力価にした。
Claims (14)
- SEQ ID NO:6に示したアミノ酸配列からなる、鳥類ニューカッスル病ウイルスHN蛋白質のエピトープであるポリペプチド。
- 前記ポリペプチドは鳥類ニューカッスル病ウイルスHN蛋白質の345番目アミノ酸から358番目アミノ酸の配列であることを特徴とする、請求項1に記載のポリペプチド。
- SEQ ID NO:6に示すアミノ酸配列を有する、変形された鳥類ニューカッスル病ウイルスHN蛋白質のエピトープを含むニューカッスル病ウイルス(寄託番号KCTC10919BP)。
- SEQ ID NO:6に示すアミノ酸配列を有する変形された鳥類ニューカッスル病ウイルスHN蛋白質のエピトープを含むニューカッスル病ウイルス(寄託番号KCTC10919BP)を含む鳥類ニューカッスル病ウイルス用ワクチン。
- 前記ワクチンは生ワクチン、弱毒化ワクチンまたは死ワクチンであることを特徴とする、請求項4に記載のワクチン。
- 抗体または抗体の断片であって、SEQ ID NO:6に示したアミノ酸配列からなる、鳥類ニューカッスル病ウイルスHN蛋白質のエピトープを特異的に認識する、抗体または抗体の断片。
- 前記抗体または抗体の断片はSEQ ID NO:6に示すアミノ酸配列を含むHN蛋白質のエピトープを含むポリペプチドを抗原にして製造され、SEQ ID NO:6に記載されたアミノ酸配列を認識することを特徴とする、請求項6に記載の抗体または抗体の断片。
- 前記抗体はモノクローナルまたはポリクローナルであることを特徴とする、請求項6に記載の抗体または抗体の断片。
- 前記鳥類は鶏、キジ、鴨、七面鳥、ウズラまたはダチョウであることを特徴とする、請求項6に記載の抗体または抗体の断片。
- 請求項6乃至9のいずれかに記載の抗体または抗体の断片を鳥類ニューカッスル病ウイルスを含む試料に反応させ、前記抗体に対する陽性反応を示す試料をニューカッスル病として判断することを含む、鳥類ニューカッスル病の診断方法。
- 請求項6乃至9のいずれかに記載の抗体または抗体の断片を鳥類ニューカッスル病ウイルスを含む試料に反応させ、鶏赤血球を反応させてニューカッスル病ウイルスによる赤血球凝集の抑制の有無を検査する前記抗体に対する赤血球凝集抑制反応法を用いてニューカッスルウイルスを探知することを含む、鳥類ニューカッスル病の診断方法。
- 前記陽性反応は、前記抗体または抗体の断片に放射線同位元素、蛍光物質、発光物質、酵素、クロモゲン及び染色物質からなる群より選択された標識物質で標識し、前記標識物質による反応結果を検出して行うことを特徴とする、請求項11に記載の鳥類ニューカッスル病の診断方法。
- 請求項6乃至9のいずれかに記載の抗体または抗体の断片を含む鳥類ニューカッスル病診断用ポリペプチドキット。
- SEQ ID NO:5に示すヌクレオチド配列を含む鳥類ニューカッスル病診断用核酸キット。
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R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |