人免疫缺陷病毒Ⅱ型(HIV-2)gag蛋白基因在毕赤酵母中的克隆表达

目的：实现HIV-2HCOgag重组蛋白的分泌表达，为研究HIV—2HCO gag蛋白结构与功能及亚单位蛋白疫苗研究打下基础。方法：将HIV-2HCOgag蛋白基因片段，与分泌型毕赤酵母表达载体pPIC9重组，构建了相应的重组表达质粒pPIC9-gag，转化GS115酵母菌，经MD／MM表型筛选、PCR扩增筛选阳性克隆，以甲醇诱导表达后进行SDS-PAGE分析及Wgtem blot证实。结果：获得了HIV—2HCOgag重组蛋白在巴斯德毕赤酵母系统中的分泌表达，表达产物可被HIV—2抗血清识别，分子量约为57kD。结论：pPIC9—gag重组质粒在甲醇营养型酵母菌中可经甲醇诱导产生HIV—2HCOgag蛋白，该蛋白具有强免疫学活性。

Expression of HIV-2 gag gene in Pichia pastoris

Objective: To cany out the secretive express of HW-2 gag protein for developing HTV-2 gag protein vaccine which is against HIV infection.Methods: Recombinant expression plasmid pPIC9-gag was constructed by insterting of HTV-2 gag into yeast expression vector pPIC-9,then transformed into GS115 cells.Positive clones were selected with MD/MM plates and confirmed by PCR.Several clones were incubated in MBGY media and induced by 0.5% methanol in BMMY media.The expression product HTV-2 gag protein was analyzed by SDS-PAGE and confirmed by Western blot.Results:The HTV-2 gag protein could be induced with methanol in Pichia pastoris.The molecular weight of the expressed protein, analyzed by SDS-PAGE,was 55 kD.The Western blot result showed that the expressed protein could be detected by HTV-2 gag specific antibody. Conclusion:The recombinant plasmid HTV-2 gag could be induced to express the HTV-2 gag protein by methanol in methanol-trophic yeast expression system.