以CotB作为融合基序在枯草芽胞杆菌芽胞表面展示雏沙门菌OmpC的研究  

Display of OmpC of Salmonella pullorum on Bacillus subtilis spores using CotB as a fusion partner

作　　者：刘明刚[1] 刘红露[1] 戴茜茜[1] 徐毓琴 张飞燕[1] 谷笑笑 潘康成[1,2] 

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]动物疫病与人类健康四川省重点实验室,四川成都611130

出　　处：《中国兽医科学》2015年第11期1159-1165,共7页Chinese Veterinary Science

基　　金：教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848)

摘　　要：本文旨在通过使用枯草芽胞杆菌芽胞衣壳蛋白CotB作为融合基序,构建表面展示雏沙门菌外膜蛋白OmpC的重组芽胞,为防控沙门菌感染提供新的思路。分别PCR扩增雏沙门菌OmpC编码基因与含自身启动子的芽胞衣壳蛋白CotB编码基因,以质粒pDG364为基础进行两者融合并构建整合型重组质粒pDG364-cotB-ompC,将融合基因整合于枯草芽胞杆菌168 amyE基因位点,通过淀粉酶活性、PCR技术、免疫荧光显微镜进行分析。结果显示,以芽胞衣壳蛋白CotB为融合基序,成功构建获得了携带cotB-ompC融合基因的整合型重组质粒pDG364-cotB-ompC,并经同源双交叉重组将其整合于枯草芽胞杆菌168基因组上;淀粉酶活性分析与PCR鉴定表明,该融合基因被成功整合于amyE基因位点,使用OmpC特异性抗体探测的免疫荧光显微镜分析检测到特异性的荧光信号。结果表明,雏沙门菌OmpC以具有生物活性的形式被成功表达并展示于枯草芽胞杆菌168的芽胞表面。The aim of this study was to construct recombinant Bacillus subtilis spores expressing the OmpC antigen of Salmonella pullorum fused to the spore coat protein CotB of B.subtilis and develop a new way for prevention and control of Salmonellainfections.The cotB with its promoter and ompCgenes were amplified by PCR from the genomic DNA of B.subtilis and S.pullorum,respectively.The cotB-ompC fusion gene was obtained by cloning ompCin frame of cotBin the integrative vector pDG364,yielding plasmid pDG364-cotB-ompC.The obtained fusion gene was integrated into B.subtilis 168 chromosome at the amyElocus by a double crossover event.Several clones were sequentially tested by PCR,amylase activity assay and immunofluorescence microscope.The results indicated that the correct insertion of cotB-ompC gene fusion at amyElocus on B.subtilis 168 chromosome and OmpC was successfully expressed and displayed on spores surface with biological activity.

关 键 词：沙门菌 枯草芽胞杆菌 芽胞表面展示 黏膜疫苗 

分 类 号：S852.612[农业科学—基础兽医学]