肠道病毒71型外壳蛋白VP2在Pichia．pastoris酵母中的表达

Expression of VP2 of Enterovirus 71 in Pichia.pastoris

利用逆转录聚合酶链式反应(RT-PCR)的方法,克隆肠道病毒71型(Entero-virus 71,EV71)SHZH03株外壳蛋白VP2基因,构建酵母分泌型表达质粒pPIC9K/VP2,经序列测定证实-αfactor信号肽和VP2的序列和阅读框正确后,用SacI酶切使之线性化,电转化法将目的基因整合到宿主菌GS115基因组上;经转化子表型筛选和PCR分析鉴定后,甲醇诱导筛选到高效表达VP2蛋白的工程菌株。用饱和硫酸铵法对重组蛋白VP2进行了较好的纯化。Western Blot分析表明,重组蛋白VP2具有较好的反应原性,从而为发展EV71快速、高效、廉价诊断试剂奠定了基础。

The genes encoding the capsid protein VP2 were amplified by RT-PCR. After confirmed by DNA sequence analysis, the VP2 was inserted into the Pichia. pastoris expression vector pPIC9K. The sequence of ORF of a factor and VP2 were confirmed by DNA sequencing with primer 5′-AOX1 and 3′- AOX1. Then the pPIC9K/VP2 were transformed into yeast GS115 by the of electro-transformation. After the PCR analysis and the screening for Mut^＋ phenotype of the transformants, multiple inserted transformants were fermented in flasks. By induction with methanol （96 h）, recombinant protein could be effectively secreted into media. SDS-PAGE analysis showed that the gene VP2 could express as 30 kDa protein as the same as the natural protein. Western Blot analysis of EV71 VP2 protein expression in Pichia. pastoris demonstrated that they had the desired immunogenicity of EV71.