JP7291973B2 - 豚流行性下痢ウイルスワクチン組成物およびその製造方法 - Google Patents
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Description
図1のように植物体で豚流行性下痢ウイルスS1タンパク質を発現させることができるように、組換え植物発現ベクターを製作した。より詳しくは、豚流行性下痢ウイルスS1タンパク質に関する遺伝子情報を確保し、ニコチアナ・ベンサミアナ(Nicotiana benthamiana)での発現に最適化した配列で遺伝子(配列番号1)を合成した。pCAMBIA1300ベクターのCaMV 35Sプロモーター遺伝子とNOSターミネーター(terminator)との間にBiP(chaperone binding protein)信号ペプチド(signal peptide)をコードするポリヌクレオチド(配列番号2)、豚流行性下痢ウイルスS1タンパク質をコードするポリヌクレオチド(配列番号1)、豚の免疫グロブリンFc断片(porcine Fc fragment;pFc)のpFc2をコードするポリヌクレオチド(配列番号3)およびHDEL(His-Asp-Glu-Leu)ペプチドをコードするポリヌクレオチド(配列番号4)を順に連結して、豚流行性下痢ウイルスS1タンパク質植物発現ベクターを製作した。
2.1.植物発現ベクター一過性発現(transient expression)
前記の実施例1で準備した植物発現ベクターをアグロバクテリア菌株LBA4404に電気穿孔法(Electroporation)を用いて形質転換させた。形質転換されたアグロバクテリアを5mlのYEP(Yeast Extract Peptone)液体培地(酵母抽出物10g、ペプトン10g、NaCl 5g、カナマイシン50mg/L、リファンピシン25mg/L)で28℃の条件で16時間振とう培養した後、1次培養液1mlを50mlの新しいYEP培地に接種して28℃の条件で6時間振とう培養した。このように培養されたアグロバクテリアは、遠心分離(7,000rpm、4℃5分)して収集した後、インフィルトレーション(Infiltration)バッファー(10mMのMES(pH5.7)、10mMのMgCl2、200μMのアセトシリンゴン)に600nmの波長でO.D.1.0の濃度でさらに懸濁させた。アグロバクテリア懸濁液は、注射針を除去した注射器を用いてニコチアナ・ベンサミアナ葉の裏面に注入する方法でアグロインフィルトレーション(Agro-infiltration)を行った。
前記の実施例2.1で準備した植物葉からタンパク質を抽出して遠心分離した後に、溶液に含まれている水溶性分画(S)にあるタンパク質とペレット(pellet;P)分画にあるタンパク質をウェスタンブロットで確認した。より詳しくは、各分画30μLをSDS試料バッファーと混合した後に加熱した。そして、10%SDS-PAGEゲルに電気泳動してサイズ別にタンパク質を分離し、分離したタンパク質をPVDF膜に移動させた後に、5%スキムミルク(skim milk)を用いてブロッキング段階を経た後に、pFc2を認識するanti-pig secondary antibodyを結合させ、ECL溶液を製造社で提供する方法によって処理して、組み換え豚流行性下痢ウイルスS1タンパク質の発現を確認した。その結果は図2に示した。
前記の実施例2.1で準備したニコチアナ・ベンサミアナ40gにタンパク質抽出溶液(50mMのTris(pH7.2)、150mMのNaCl、0.1%Triton X-100、1Xタンパク質加水分解酵素阻害剤(protease inhibitor))200mLを添加し、ブレンダで組織を破砕した後、13,000rpmで20分間4℃で遠心分離してタンパク質抽出液を回収した。発現した組み換え豚流行性下痢ウイルスタンパク質の分離精製のために、protein A-sepharoseレジンが充填されたカラムで親和クロマトグラフィーを実施した。カラムにレジンを5mL充填した後、洗浄溶液(50mMのTris(pH7.2)、150mMのNaCl)50mLで平衡化させた。回収したタンパク質抽出液をカラムに適用した後、洗浄溶液100mLを流してレジンを洗浄し、溶出溶液(0.1Mのsodium citrate(pH 3.0)、150mMのNaCl)で組み換えタンパク質を溶出し、中和溶液(1MのTris(pH9.0))を添加してタンパク質溶出液を中和させた。組み換えタンパク質が含まれた溶出溶液は、30kDの大きさのフィルターを使用して生理食塩水(PBS)溶液に交換および濃縮を実施して、分離精製された組み換え豚流行性下痢ウイルスタンパク質を獲得した。分離精製されたタンパク質は、タンパク質電気泳動(SDS-PAGE)後、クマシー染色法(coomassie staining)を通じて確認した(図3)。
組み換え豚流行性下痢ウイルスS1タンパク質抗原が生体内で抗体を誘導して免疫原性を有するかを確認するために、各グループ当たり5匹ずつの6週齢の雌ギニアピッグを用いて実験を進めた。より詳しくは、陰性対照群は、生理食塩水を投与し、陽性対照群は、市販の豚流行性下痢ワクチン(中央ワクチン)を投与し、実験群は、組み換え豚流行性下痢ウイルスS1タンパク質を1回当たり150μgずつ3週間隔で2回皮下で投与した。抗原の投与時には、同量のIMS1313 adjuvant(SEPPIC)を混ぜて注射した。血液は、抗原の投与前と2次投与後2週目後に頚静脈または心臓から採取後、血清を分離して、-20℃に冷凍保管した。各血清で豚流行性下痢ウイルスS1タンパク質に対する特異抗体の生成は、His tagが融合したPEDV-S1組み換えタンパク質がコートされたエライザ(ELISA)プレートを用いて確認し、その結果は、図4に示した。
前記の実施例4で準備した血清で組み換え抗原タンパク質の投与で形成された血中抗体が豚流行性下痢ウイルス中和能を有するかを確認した。より詳しくは、中和抗体の試験には、PEDV QIAP1401 strainウイルスとVero cellを感染宿主として使用した。採血された血液の血清を分離して57℃で30分間不活性化した後、10ug/mlのtrypsinが含有されたMEM(Minimum Essential Media Eagle)培地で2倍希釈した。各希釈血清に316 TCID50/0.1mlのPEDVウイルスを同量混合して、37℃で90分間中和させた。培養が終わる直前に、96ウェルプレートにモノレイヤ状態で準備したVero cellをPBSで3回洗浄後、血清-ウイルス混合液100μlを添加後、37℃で2時間培養した。培養が終わった後、2μg/mlのトリプシンが添加された培地を100μl添加後、37℃で細胞変性効果(CPE)が現れるまで3日~5日間培養した。中和抗体の力価は、CPEが現れる直前にウェルの血清希釈倍数を顕微鏡下で観察して測定した(表1)。
1.1.散剤の製造
Claims (13)
- 配列番号5で表されるアミノ酸配列からなる豚流行性下痢ウイルスタンパク質を有効成分として含む、豚流行性下痢ウイルスワクチン組成物。
- 配列番号5で表されるアミノ酸配列からなる豚流行性下痢ウイルスタンパク質を有効成分として含む、豚流行性下痢の予防または治療用飼料組成物。
- 請求項1に記載のワクチン組成物をヒトを除いた動物に投与することによって、豚流行性下痢を予防または治療する方法。
- 配列番号1で表されるポリヌクレオチド配列を含む、配列番号5で表されるアミノ酸配列からなる豚流行性下痢ウイルスタンパク質発現用ベクター。
- 前記ベクターは、図1に記載された構造を有することを特徴とする請求項4に記載のベクター。
- 前記ベクターは、小胞体信号ペプチド、Fc断片およびBiP(Chaperone binding protein)よりなる群から選ばれた1つ以上をコードする遺伝子をさらに含むことを特徴とする請求項5に記載のベクター。
- 前記小胞体信号ペプチドは、KDEL、HDEL、SEKDEL、KHEDL、KEEL、およびSEHEDLで表されるペプチド配列よりなる群から選ばれた1つであることを特徴とする請求項6に記載のベクター。
- 請求項4~7のいずれか一項に記載のベクターで形質転換された、形質転換体。
- 前記形質転換体は、植物であることを特徴とする請求項8に記載の形質転換体。
- (a)請求項8に記載の形質転換体を培養する段階と、
(b)前記形質転換体または培養液から豚流行性下痢ウイルスタンパク質を分離および精製する段階と、を含む、配列番号5で表されるアミノ酸配列からなる豚流行性下痢ウイルスタンパク質の生産方法。 - 前記段階(b)の精製は、水溶性分画を使用して精製することを特徴とする請求項10に記載のタンパク質の生産方法。
- 配列番号5で表されるアミノ酸配列からなる豚流行性下痢ウイルスタンパク質を有効成分として含む組成物を用いることを含む、豚流行性下痢の予防または治療方法(ヒトを対象とするものを除く)。
- 配列番号5で表されるアミノ酸配列からなる豚流行性下痢ウイルスタンパク質の豚流行性下痢ワクチンまたは薬剤の製造のための使用。
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