枯草杆菌芽孢表面展示猪流行性腹泻病毒S蛋白保护性抗原片段研究

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• 英文论文题名：The study of the partial protective gene of Porcine epidemic diarrhea Virus S protein displayed on the surface of Bacillus subtilis spore
• 论文作者：戴茜茜 ; 谷笑笑 ; 张飞燕 ; 王振华 ; 张娇 ; 冯杰 ; 潘康成
• 英文论文作者：Xixi Dai ; Zhenhua Wang ; Xiaoxiao Gu ; Jiao Zhang ; Jie Feng ; Kangcheng Pan ; Animal Microecology Institute ; College of Veterinary Medicine ; Sichuan Agricultural University ; Chengdu agricultural science and technology Career Academy
• 年：2016
• 作者机构：四川农业大学动物医学院动物微生态研究中心;成都农业科技职业学院;
• 论文关键词：猪流行性腹泻病毒 ; 枯草芽孢杆菌 ; 芽孢表面展示 ; cotB ; S蛋白
• 英文论文关键词：Porcine epidemic diarrhea ; Bacillus subtilis ; spore surface display ; cotB ; S protein
• 会议召开时间：2016-11-04
• 会议录名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会论文集
• 语种：中文
• 分类号：S852.651
• 学会代码：ZGXJ
• 会议名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会
• 会议地点：中国山东青岛
• 主办单位：中国畜牧兽医学会动物微生态学分会
• 学会名称：中国畜牧兽医学会
• 页数：1
• 文件大小：84k
• 原文格式：O
• 会议级别：全国

摘要

本研究将猪流行性腹泻病毒(PEDV)S蛋白保护性抗原片段COE_1与枯草杆菌芽孢外层衣壳蛋白基因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孢。通过将cotB基因与COE1编码序列进行重组,构建融合表达cotB-COE_1的整合型重组质粒,重组质粒与枯草芽孢杆菌淀粉酶基因amyE双交叉,获得重组枯草芽孢杆菌,对重组枯草芽孢杆菌进行淀粉酶活性分析、PCR扩增鉴定,提取重组芽孢衣壳总蛋白进行Westernblot分析及对重组芽孢进行免疫荧光显微镜观察。通过淀粉酶活性分析与PCR鉴定结果表明该融合基因成功整合于枯草芽孢杆菌染色体上;Western blot分析结果在67.78kDa处获得一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重组芽孢具有特异性荧光信号。结果表明PEDV S蛋白保护性抗原片段COE_1成功地展示于枯草杆菌芽孢表面。
The aim of this study was to construct a gene fusion between protective antigen segment COEi of Porcine epidemic diarrhea virus(PEDV) S protein and Bacillus subtilis spore coat gene cotB,finally constructed a recombinant Bacillus subtilis with the ability to display the protective antigen fragments of protein S on Bacillus subtilis spore surface.We recombinanted cotB-COEi gene under the control of the cotB promoter and constructed a integrative recombinant plasmid carrying cotB-COE\,integrative recombinant plasmid was transformed into Bacillus subtilis amylase gene amyE by double cross-over and the recombinant Bacillus subtilis was produced,amylase activity and PCR were used to monitor the recombinant Bacillus subtilis,recombinant bacillus coat proteins was extracted for Western blot analysis and the recombinant Bacillus subtilis spores was observed under the fluorescence immunoassay.The insertion of cotB-COEi in the amyE locus were confirmed by amylase activity analysis and PCR;Western blot analysis showed that a specific band about 67.78 kDa was detected in the recombinant spores extracts using PEDV antibodies and significant rad fluorescence was observed on recombinant spores surface under fluorescent microcopy.This result suggested that the partial protective gene COEi of S protein of PEDV was displayed on Bacillus subtilis spore surface successfully.

引文

[1]PENSAERT M B,BOUCK P D.A new coronavirus-like particle associated with diarrhea in swine[J].Arch Virol,1978,58(3):243-247.
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