猪流行性腹泻病毒M蛋白的原核表达  

Prokaryotic expression of porcine epidemic diarrhea virus M protein

作　　者：刘梦茜[1] 李春燕[1] 陈仕怡 袁晓琴[1] 星东 王全溪[1] LIU Mengxi LI Chunyan CHEN Shiyi YUAN Xiaoqin XING Don g WANG Quanxi(College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,Chin)

机构地区：[1]福建农林大学动物科学学院,福建福州350002

出　　处：《福建农林大学学报（自然科学版）》2017年第3期299-304,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建农林大学科学发展基金资助项目(KF2015094)

摘　　要：本试验设计了M基因的特异性引物,扩增了M基因,成功构建了重组质粒,并转化至Escherichia coli BL21(DE3)感受态细胞中,优化了异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达条件.结果表明,重组表达的融合蛋白Pet-32a-M大小约为43 ku,与预期大小相符.十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表明,M蛋白为包涵体蛋白,且在IPTG浓度为0.8 mmol·L-1,温度为37℃,诱导时间为8 h的条件下,蛋白表达效果最佳.蛋白免疫印迹检测结果进一步显示,重组蛋白Pet-32a-M在体外可以被成功表达.To increase the expression level of M protein which is a candidate antigen for porcine epidemic diarrhea virus, specific primer for M gene was designed and amplified. The positive recombinant plasmid was identified and transformed into Escherichia coli BL21（DE3）competent cell, and followed by being induced by isopropyl β-D-thiogalactoside（IPTG）. Then the inducible expression system was optimized in terms of IPTG concentration, expression time and temperature. Results showed that the recombinant protein was successfully expressed, with the molecular weight being about 43 ku. SDS-Polyacrylamide gelelectrophoresis（SDS-PAGE）analysis indicated that M protein is an inclusion body protein. The optimal induction conditions were to add 0.8 mmol·L^-1 IPTG and be induced at 37 ℃ for 6 h. In addition, Western-blot contfirmed that the recombinant plasmid Pet-32a-M was transformed into E.coli BL21（DE3）competent cell and successfully expressed in vitro.

关 键 词：猪流行性腹泻病毒 M蛋白 原核表达 

分 类 号：S852.651[农业科学—基础兽医学]