枯草芽胞杆菌芽胞表面展示猪流行性腹泻病毒S蛋白保护性抗原片段的研究  

Study on the partial protective gene of porcine epidemic diarrhea virus S protein displayed on the surface of Bacillus subtilis spore

作　　者：戴茜茜[1] 王振华[2] 谷笑笑 张娇[1] 冯杰[1] 潘康成[1] 

机构地区：[1]四川农业大学动物医学院动物微生态研究中心,四川成都611130 [2]成都农业科技职业学院,四川成都611100

出　　处：《中国兽医科学》2016年第1期38-45,共8页Chinese Veterinary Science

基　　金：教育部"长江学者和创新团队发展计划"创新团队资助项目(IRT0848);"四川省高等学校科技创新团队"资助项目

摘　　要：将猪流行性腹泻病毒(PEDV)S蛋白保护性抗原片段COE1与枯草芽胞杆菌芽胞外层衣壳蛋白基因cotB进行融合,构建表面展示S蛋白保护性抗原片段的重组芽胞。通过将cotB基因与COE1编码序列进行重组,构建融合表达cotB-COE1的整合型重组质粒,重组质粒与枯草芽胞杆菌淀粉酶基因amyE双交叉,获得重组枯草芽胞杆菌。对重组枯草芽胞杆菌进行淀粉酶活性分析、PCR鉴定,提取重组芽胞衣壳总蛋白进行Western-blot分析及对重组芽胞进行免疫荧光显微镜观察。淀粉酶活性分析与PCR鉴定结果表明,该融合基因成功整合于枯草芽胞杆菌染色体上;Western-blot结果显示,在67.78ku处获得1条特异性的蛋白杂交带;使用生物素标记抗体免疫荧光显微镜观察,可见重组芽胞具有特异性荧光信号。结果表明,PEDV S蛋白保护性抗原片段COE1成功地展示于枯草芽胞杆菌芽胞表面。The aim of this study was to construct a gene fusion between protective antigen segment COE1 of porcine epidemic diarrhea virus（PEDV）S protein and Bacillus subtilis spore coat gene cotB,finally constructed a recombinant B.subtilis with the ability to display the protective antigen fragments of S protein on B.subtilis spore surface.We recombinanted cotB-COE1 gene under the control of the cotBpromoter and constructed a integrative recombinant plasmid carrying cotB-COE1.Integrative recombinant plasmid was transformed into amylase gene amyE of B.subtilis by double cross-over and the recombinant B.subtilis was produced,amylase activity and PCR were used to monitor the recombinant B.subtilis,recombinant bacillus coat protein was extracted for Western-blot analysis and the recombinant B.subtilis spores were observed under the fluorescence immunoassay.The insertion of cotB-COE1 in the amyE locus was confirmed by amylase activity analysis and PCR.Western-blot analysis showed that a specific band about 67.78 ku was detected in the recombinant spores extracts using PEDV antibodies and significant red fluorescence signal was observed on recombinant spores surface under fluorescent microscope.This result suggested that the partial protective gene COE1 of S protein of PEDV was displayed on B.subtilis spore surface successfully.

关 键 词：猪流行性腹泻病毒 枯草芽胞杆菌 芽胞表面展示 cotB S蛋白 

分 类 号：S852.659.6[农业科学—基础兽医学]