肠道病毒71型外壳蛋白VP1在Pichia pastoris酵母中的表达

Expression of Capsid Protein VP1 of Enterovirus Type 71 in Pichia pastoris

利用逆转录聚合酶链式反应(RT PCR)扩增肠道病毒71型(EV71)外壳蛋白VP1基因,经序列测定证实后,构建重组表达质粒pPIC9K/VP1,转化Pichiapastoris酵母宿主菌GS115,甲醇诱导表达。SDS PAGE分析显示:表达产物的分子量约为34kD,与天然VP1大小一致。凝胶薄层扫描分析显示:目的蛋白表达量占培养上清总蛋白的60%以上。ELISA实验表明,重组蛋白VP1具有较好的抗原性。使用饱和硫酸铵分级沉淀法初步纯化的表达产物,能够较特异性地与EV71感染者血清中的抗体产生反应,而且与抗柯萨奇病毒A16特异性抗体不产生反应。通过利用表达产物作为抗原,对156份血清的检测初步证实,重组蛋白VP1可以作为检测EV71感染的的检测用抗原。

The gene encoding the entire capsid protein VP1 of enterovirus type 71(EV71) was amplified by RTPCR.After confirmed by sequence analysis,VP1 was expressed in Pichia pastoris (GS115).SDSPAGE showed that the geneVP1 could express product of 34kD as same as the natural protein.The density scanning of the SDSPAGE gel revealed that the target protein accounted for 65% of the total protein in the supernatant.ELISA analysis of the culture supernatant confirmed it had the desired immunogenicity of EV71.After concentrated by ammonium sulfate precipitation,the recombinant protein VP1 could specifically react with EV71 patients' sera,but not with antiCox A16 serum.By testing 156 sera with recombinant protein VP1 as antigen,it revealed that recombinant VP1 of EV71 may be an effective mean for determining EV71infection of different disease phases.