鼠伤寒沙门氏菌鞭毛蛋白FliC的原核表达及纯化  

Expression and Purification of Fusion Protein of S.typhimurium FliC

作　　者：王鹤[1] 梁宏儒[1] 胡旭[1] 赵达[1] 姜东君[1] 尹辉[1] 高佳滨[1] 陈为宏[1] 崔玉东[2] 朱战波[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319 [2]黑龙江八一农垦大学生命科学技术学院

出　　处：《黑龙江八一农垦大学学报》2012年第5期50-54,共5页journal of heilongjiang bayi agricultural university

基　　金：黑龙江省研究生创新科研项目(YJSCX2011-279HLJ);国家自然科学基金项目(31072120)

摘　　要：对鼠伤寒沙门氏菌的鞭毛蛋白FliC的基因进行克隆、鉴定和原核表达,为鞭毛蛋白佐剂作用的研究奠定基础。以鼠伤寒沙门氏菌8014菌株基因组DNA为模板,PCR扩增Flic基因,产物与T载体连接,经测序鉴定正确后与表达载体pQE30(+)连接构建重组表达质粒Flic-pQE30,将此重组质粒转化入表达宿主E.coli XL1-Blue菌株内抽提质粒,酶切鉴定正确后对转化菌株以IPTG进行诱导后,表达产物经镍离子亲和层析柱纯化后,进行SDS-PAGE和Western blot分析。测序结果显示,FliC为完整的编码区基因1 485 bp,编码495个氨基酸残基,蛋白相对分子质量约为56 kDa。经SDS-PAGE分析表明,以37℃、1 mM的IPTG诱导5 h,表达效果最好,诱导产物是与理论值相符的56 kDa的融合蛋白。经western-blotting检测,表达的重组蛋白FliC可与小鼠抗鼠伤寒沙门氏菌全菌体多抗血清反应得到清晰的目的条带,表明表达的重组蛋白具有良好的反应原性。成功进行了鼠伤寒沙门氏菌鞭毛蛋白Flic基因的克隆表达,为进一步研究其免疫佐剂作用奠定了基础。The purpose of this study was to clone, express and identify FliC protein of Salmonella typhimurium in order to provide the foundation for the study of adjuvant effects of FliC. The gene encoding protein FliC was amplified from the genomic DNA of S.typhimurium 8014 by using PCR technique. The amplified product was cloned into pMD18-T. Plasmids containing the right insert were sequenced to confirm its identity,and then cloned into expression vector pQE30 （＋）. The constructed recombinant plasmid Flic-pQE30 was transformed to E.cali XL1-Blue and induced with IPTG, and the expressed product was identified by SDS-PAGE and Western blot. Sequence analysis showed that the full coding length of Flic was 1 485 bp,which could encode 495 amino acid residues with a molecular mass of 56 kD.The SDS-PAGE electrophoresis analysis showed that the best expression was induced in 5 hours by 37 ~C and 1 mM IFl＇G,under which a relative molecular weight of 56 kD recombinant protein FliC was produced. The recombinant FliC showed specific reaction with sera of rats immunized with S.typhimurium by Western blotting,which showed that the recombinant protein has well reactogenicity. The prokaryotic expression vector for fliC gene was constructed successfully,and fusion protein was expressed in E.coli XLl-Blue,and lay the foundation for the study of adjuvant effects of FliC.

关 键 词：鼠伤寒沙门氏菌 鞭毛蛋白 克隆 表达与鉴定 

分 类 号：S855.12[农业科学—临床兽医学]