肠道病毒71型VP3结构蛋白的原核表达

Optimization of prokaryotic expression of enterovirus 71 VP3 capsid protein

目的利用大肠杆菌原核表达系统优化表达纯化肠道病毒71型(EV71)VP3结构蛋白,为后续单克隆抗体制备及检测试剂盒研发提供前期基础。方法采用PCR方法扩增EV71病毒VP3基因,将其插入表达载体pET28a(+),构建pET28a-VP3重组质粒,转化大肠杆菌BL21(DE3)菌株,分别在25℃、37℃下经IPTG诱导表达,重组表达的蛋白产物经凝胶电泳初步分析,比较不同温度诱导表达的蛋白产物。结果成功构建pET28a-VP3重组质粒,不同温度下诱导表达的蛋白产物在30.5 k Da左右位置均出现目的条带;37℃下诱导表达的蛋白超声破碎并离心后,目的蛋白基本位于沉淀中,而25℃诱导表达的蛋白产物有少量目的蛋白溶解于上清液中。结论在25℃或37℃下均能利用大肠杆菌原核表达系统有效表达EV71病毒VP3蛋白;37℃诱导时蛋白可融性表达低,目的蛋白获取效率较高。

Objective To express VP3 capsid protein of enterovirus 71 by using Escherichia coli prokaryotic expression system. Methods VP3 gene was amplified by PCR before inserted into p ET28a（＋） plasmid. Then the plasmid p ET28a-VP3 was transformed and expressed in the Escherichia coli BL21 strain at 25 ℃ or 37 ℃. Finally the protein was analyzed by SDS-PAGE gel electrophoresis. Results The p ET28a-VP3 plasmid was successfully constructed,and the EV71VP3 protein was expressed. Supernatant of the production after ultrasonication and centrifugation got a little VP3 protein.Conclusion The EV71 VP3 protein was expressed. Expression at 25 ℃ may lead to the dissolution of the recombinant protein.