Development of Discriminating Diagnostic Reagents of Classic Swine Fever Virus I. Large-Scale Production of Viral Proteins

Abstract

計畫目標: 應用酵母菌Pichia pastoris表現系統大量生產豬瘟病毒各結構蛋白( 包括醣蛋白E0、E1與E2及核蛋白C )以提供後續建立ELISA鑑別診斷方法之材料.架構( 重要工作項目 ): 1.豬瘟病毒結構蛋白基因Pichia重組表現質體之構築: 應用聚合酵素連鎖反應( PCR )方法分別針對豬瘟病毒E0、E1與E2及核蛋白C各基因片段進行增幅後選殖於pCRII-TOPO載體, 並經核酸定序確認所選殖基因片段的正確性.接著分別純化出各基因片段並轉殖於酵母菌pPICZα表現載體上, 完成各豬瘟病毒結構蛋白基因Pichia重組表現質體之構築.2.重組P.pastoris酵母菌株之篩選: 各重組質體分別經轉形作用( transformation )送入P.pastoris細胞後培養於含100μg/ml Zeocin之培養基, 待2-4天後分別收集具抗藥性之菌落.萃取各菌株之染色體DNA進行PCR快速檢測以確定是否含有各特定豬瘟病毒基因.而各菌株經以甲醇( 最終濃度為0.5% )進行誘導表現後不同時間分別收取菌液, 以蛋白質電泳分析測定其表現情形.進一步再利用抗豬瘟病毒之免疫血清進行西方轉漬分析( western immunoblotting )以挑選出能大量生產豬瘟病毒蛋白且具正確抗原性的重組P.pastoris菌株.3.重組P.pastoris大規模發酵培養: 選擇表現特性最佳之各重組菌株先經隔夜培養於200ml培養液後接種於裝有二公升培養液之發酵槽, 並設定最適當之溫度、攪拌速率及空氣進氣量等條件進行發酵培養.於發酵培養起始即加入甲醇並持續維持其濃度為0.5%以誘導豬瘟病毒蛋白的大量表現.4.豬瘟病毒蛋白純化及其抗原性分析: 分泌性蛋白之純化可直接收集培養上清液, 並加入飽和硫酸銨溶液來沈澱出蛋白質或以超過濾( ultrafiltration )方式達到濃縮蛋白質含量.若是細胞內蛋白產物則先利用玻璃珠微粒將酵母菌體打破後再收集細胞溶解上清液進行純化.純化之各病毒蛋白表現產物分別免疫小白鼠進行免疫血清之製備, 並進而利用各血清學診斷方法檢測這些表現蛋白所誘發產生抗體之力價與特異性.預期效益: 豬瘟病毒醣蛋白產物可進一步開發成為次單位標識疫苗, 而核蛋白產物則可應用做為鑑別診斷試劑, 有利於豬瘟撲滅計畫之進行.Classical swine fever virus ( CSFV )is the causative agent of the highly contagious infection of swine-classical swine fever ( hog cholera ).The virion contains four structural proteins: E0, E1and E2glycoproteins as well as the nucleocapsid ( core )protein C.The purpose of this study is to produce large amounts of CSFV structural proteins by the yeast Pichia pastoris eukaryotic expression system and for further developing CSFV subunit marker vaccine and companion discriminating test.The viral structural genes are amplified and cloned by the polymerase chain reaction ( PCR )techniques and then subcloned onto the Pichia expression vector to construct recombinant expression plasmids containing the CSFV E0, E1, E2and C genes respectively.These recombinant expression plasmids are transformed into yeast P.pastrois competent cells and the stable expression recombinant yeast strains are selected and characterized.A large-scale fermentation culture will be set-up for the preparation of large amounts of expressed CSFV proteins.The expression products from the fermentation culture will be purified and used as antigens to immunize mice in order to assess the specificity of the antibodies induced by those expressed proteins.The results obtained from this project will provide the basis for the development of diagnostic reagents for evaluation and differentiation of the immune reactions induced by CSFV subunit marker vaccine