猪流行性腹泻病毒CH/S株M蛋白全长的原核表达及其反应原性分析  

Expression of the whole M protein of porcine epidemic diarrhea virus CH/S strain in Escherichia coliand analysis of its reactogenicity

作　　者：张志榜[1] 陈建飞[1] 时洪艳[1] 陈小金[1] 冯力[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室猪传染病研究室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2011年第1期31-35,共5页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2006BAD06A07)

摘　　要：根据猪流行性腹泻病毒(PEDV)CH/S株M蛋白的基因组序列,设计了1对引物,采用RT-PCR方法扩增出M基因,将其克隆至原核表达载体pMXB10,构建了重组质粒pMXB-M,经测序鉴定正确后转化感受态细胞BL21(DE3),并进行了IPTG诱导表达。结果显示,重组菌pMXB-M表达的融合蛋白MBP-M-CBD(rM)的分子质量约为95ku,在IPTG浓度为0.8mmol/L、诱导时间为6h时,重组菌的表达效果最好,重组蛋白以包涵体的形式存在于菌体中。Western-blot结果显示,rM能与兔抗PEDV多克隆抗血清及PEDV M蛋白单克隆抗体发生特异的免疫印迹反应,表明原核表达的rM蛋白具有良好的反应原性,这为进一步开展PEDV M蛋白的研究奠定了基础。One pair of primers was designed according to the published sequences of M gene of porcine epidemic diarrhea virus（PEDV） CH/S strain.The M gene was amplified with the pair of primers by RT-PCR and cloned into the prokaryotic expression vector pMXB10 to construct a recombinant plasmid pMXB-M.After sequencing,the recombinant was transformed into Escherichia coli BL21（DE3） competent cells.The transformed bacteria induced by IPTG produced a fusion protein（MBP-M-CBD） of 95 ku.In result,inducing with 0.8 mmol/L IPTG for 6 h was the best conditions for expression of M protein.The recombinant protein expressed in the bacteria was in the form of inclusion bodies.The recombinant protein could react with the rabbit anti-PEDV serum and the monoclonal antibody against M protein of PEDV in Wes-tern-blot tests,indicating that the expressed protein possessed reactogenicity.The result laid the foundation for further studies of PEDV M protein.

关 键 词：猪流行性腹泻病毒 M蛋白 原核表达 免疫印迹 

分 类 号：S852.659.6[农业科学—基础兽医学]