肠道病毒71型重组VP1蛋白的原核表达及初步鉴定

目的利用大肠杆菌原核表达系统表达并纯化肠道病毒71型(EV71)VP1蛋白,并对重组蛋白功能进行初步鉴定。方法 PCR扩增EV71VP1全长基因,在测序正确的基础上,将其插入表达载体pET28a(+),构建表达质粒pET28a(+)/VP1,转化表达菌BL21(DE3),IPTG诱导表达,利用Ni 2+亲和层析柱对重组蛋白进行纯化,经ELISA和Western Blot,验证EV71VP1的抗原性。结果成功构建pET28a(+)/VP1重组质粒,经大肠杆菌表达、纯化获得分子量约为43kDa的融合蛋白,经ELISA和Western Blot,结果显示该蛋白有良好的抗原性。结论实现了肠道病毒7l型VP1蛋白的原核高效表达,为肠道病毒71型诊断试剂和疫苗的研究奠定基础。

Prokaryotic expression and preliminary characterization of recombinant enterovirus71 VP1 protein

Objective To express the structural protein VP1 of enterovirus71 in E. coli, and characterize the recombinant protein function. Methods VP1 gene was amplified by PCR and cloned into pET28a （＋） vector after sequencing. The pET28a （＋）/VP1 recombinant plasmid was transformed into E. coli host BL21 （DE3）, and was induced by IPTG. The recombinant protein was purified by metal （Ni2＋） chelating affinity chromatography, and its immunoreactivity was tested by ELISA and Western Blot. Results The recombinant plasmid was constructed and the protein with a molecular weight of 43kDa was ex pressed and purified. The recombinant VP1 can be found by EV71 positive sera with high affinity and specificity in ELISA and Western Blot format. Conclusion The capsid protein VP1 of EV71 was expressed successfully,which provides foundation for the VPlbased vaccine and diagnostic reagent research for EV71.