汉坦病毒76-118株G2基因的克隆及其在甲基营养型酵母中的表达

Cloning of G2 Gene of Hantavirus Strain 76-188 and Expression of It in Pichia.pastoris

目的 获得汉坦病毒囊膜蛋白G2体外表达产物。方法 应用RT -PCR方法扩增汉坦病毒 76 -118株M片段编码的G2蛋白基因 ,并将扩增产物分别插入pPIC9K和pHIL -S1载体 ,命名为pPIC9K -G2和pHIL -S1-G2 ,分别与酵母的α因子信号肽和PH0 1信号肽 3’端融合 ,处于醇氧化物酶 (AOX1)启动子下游。这 2个载体经线性化后分别转化甲基营养型酵母菌株GS115和SMD116 8,筛选后分别得到His+ Muts 转化子和His+ Mut+ 转化子 ,诱导表达后 ,用Dot ELISA检测。结果 Dot-ELISA检测为阳性。结论 成功地表达了汉坦病毒G2囊膜蛋白 ,为进一步研究其结构功能以及生物学特性奠定了良好基础。

Objective To express the envelope protein G2 of Hantan virus in vitro.Methods The G2 protein gene encoded by a part of M fragment of Hantan virus strain 76-118 was amplified by RT-PCR from cDNA and inserted into vectors pPTC9K and pHIL-SI separately.The constructed plasmids pPIC9K-G2 and pHIL-S1-G2 were fused with the 3'-terminals of factor α and PHO1 signal sequeces both under the control of AOX1 promoter respectively.The 2 expression plasmids were linearized and transformed to methylotrophic yeast trains GS115 and SMD 1168,then His +Muts and His +Mut + transformants were screened out respectively and expressed under the induction of methanol.The expressed products were detected by Dot-ELISA.Results Dot-ELISA proved that G2 envelope protein was successfully expressed.Conclusion The successful expression of G2 envelope protein of Hantavirus laid a good foundation of further study on its structure,function and biological characteristics