猪葡萄球菌ExhB基因在E．coli中的表达及生物学活性分析  

Cloning and expression of the Staphylococcus hyicus ExhB gene in E.coli and its function analysis

作　　者：杨彩娟[1,2] 张曦[1,2] 蒋智勇[2] 陈德坤[1] 宋长绪[2] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]广东省农业科学院兽医研究所,广东广州510640

出　　处：《中国兽医学报》2009年第4期399-402,450,共5页Chinese Journal of Veterinary Science

基　　金：国家食品安全重大专项(2001BA804A31);广东省农业科技攻关资助项目(2003B21404、20052490100和2006B20301013);广东省自然科学基金(04002083);广东省自然基金重点项目(06105311)

摘　　要：以猪葡萄球菌基因组DNA为模板,利用PCR方法扩增ExhB基因,克隆测序鉴定后,插入质粒pET32a,构建重组表达载体pET32a-ExhB,通过大肠杆菌BL21表达重组融合蛋白,利用Ni亲和层析的方法纯化重组的融合蛋白,并用纯化的蛋白攻毒裸鼠,鉴定其生物学活性。结果显示,扩增出了猪葡萄球菌ExhB基因并纯化出了重组的融合蛋白,SDS-PAGE分析表明,融合蛋白的相对分子质量约为45 000,主要以包涵体形式存在;通过ELISA和琼脂扩散试验表明,所表达的包涵体蛋白可以与用菌体制得的多克隆抗血清发生特异性结合;用复性的蛋白在裸鼠身上攻毒后可以产生皮炎病变。结果表明,成功的表达了ExhB基因的重组融合蛋白,并且鉴定出重组蛋白可与猪葡萄球菌阳性血清发生特异性免疫反应和重组蛋白在裸鼠身上的致病性,为进一步研究猪葡萄球菌的致病机理及建立该菌完备准确的防治和检测方法奠定基础。Cloning, expression Staphylococcus hyicus ExhB gene which can cause exudative epidermitis in the piglet and identification the immunity of the protein encoded by ExhB gene,and then the analysis of the biological function of the expression products. The Staphylococcus hyicus genomic DNA was PCR amplified by the primerl and primer 2. We can get the ExhB gene in this way. After this gene was identified by clone and sequence analysis,it was cloned in the plasmid vector pET32a. The recombinant bacteria was transferred into BL21 in order to realize its expression. The protein was purified by affinity chromatography. We also inject the recombinant protein into the nude mice and identify its biological activity. Amplified the S. hyicus ExhB gene and purified the recombinant protein. SDS-PAGE showed the molecular weight of recombinant protein was 45 000. Most of the protein was existed in in-cluding body. The ELISA and agar pervasion experiment indicate that the recombinant protein could combination with the polyclonal mice antibodies immunized by the S. hyicus. The recombinant protein can cause histological change in nude mice. Successfully construct the recombinant protein. The protein could react with the polyclonal serum. The nude mice are susceptible to the protein too. This experiment can be the basis for the more deep research such as the study of the pathogenesis of S. hyicus and the exactly treatment and prevention method.

关 键 词：猪葡萄球菌 ExhB基因 基因表达 生物活性 

分 类 号：S852.611[农业科学—基础兽医学]