手足口病主要病原EV71衣壳蛋白的基因克隆与表达
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【摘要】 目的：构建EV71基因的原核表达载体，并在原核细胞中进行表达与纯化。方法：根据已知EV71全长核酸序列设计引物，用PCR方法扩增VP1、VP2、VP3和VP4四个基因片段，对目的基因进行酶切并克隆至pET-14b原核表达载体中，转化大肠杆菌BL21（DE3）感受态细胞，IPTG诱导表达;所获得的不可溶性蛋白经尿素裂解超声，亲和层析纯化，用SDS-PAGE检测表达产物。结果：测序证实原核重组质粒构建成功;可表达高纯度的四个蛋白，且蛋白大小与预计值相符。结论：成功构建了手足口病主要致病原肠道病毒71型（EV71）的四个衣壳蛋白VP1、VP2、VP3和VP4的原核表达载体，并纯化得到高纯度的四个蛋白，为进一步研究手足口病致病机制奠定了基础。
【关键词】 EV71; 基因克隆; 蛋白表达
Gene Clone and Expression of Capsid Proteins of EV71-the Main Pathogen of HFMD/LIANG Jie，LIU Aihua.//Medical Innovation of China，2019，16（06）：00-008
【Abstract】 Objective：To construct a prokaryotic expression vector of EV71 gene and have gene expression and purification in prokaryotic cells.Method：According to the full-length gene sequence of EV71 reported in GenBank，the primers were designed by using DNA Star software，based on which VP1-VP4 genes were amplified by PCR and inserted into prokaryotic expression vector pET-14b respectively.The constructed recombinant plasmid pET-14b-VPs were transformed to E.coli of BL21 strain for recombinant protein expression under induction of IPTG.The insoluble proteins were lysed in urea by supersonic，and the supernatant was collected for the purification with Ni2+-affinity chromatography，followed by SDS-PAGE.Result：Restrict enzyme digestion analysis and DNA sequencing proved that recombinant plasmids pET-14b-VP1-4 were constructed correctly.We found that the VP recombinant ...
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