人乳头瘤病毒16型L1/E7嵌合基因的克隆及其在毕赤酵母中的表达

目的克隆人乳头状瘤病毒16型(HPV16)L1/E7嵌合蛋白基因,并在毕赤酵母中表达。方法PCR扩增HPV16L1/E7基因,并克隆至毕赤酵母表达载体pPIC-3·5K和pPIC-9K中,构建含HPV16L1/E7基因的pPIC3·5K-HPV16L1/E7和pPIC9K-HPV16L1/E7重组表达载体,经测序证实插入的外源基因读码框正确后,分别转入GS115和KM71菌株中,筛选阳性转化菌株,经PCR鉴定,甲醇诱导表达HPV16L1/E7嵌合蛋白,经SDS-PAGE和Westernblot鉴定,电镜观察,并经离心法纯化VLPs。结果HPV16L1/E7基因已成功插入pPIC-3·5K和pPIC-9K载体中,共获得6株KM71HPV16L1/E7-pPIC3·5K、5株GS115HPV16L1/E7-pPIC3·5K、1株KM71HPV16L1/E7-pPIC9K和2株GS115HPV16L1/E7-pPIC9K阳性转化菌株,并可表达相对分子质量约为69000的蛋白,与预期值一致。表达量约为0·35~1·04mg/ml。Westernblot显示该蛋白可与抗-HPV16L1蛋白和抗-HPV16E7蛋白的单克隆抗体特异性结合。在透射电镜下可观察到VLPs的形成,其形态与HPV16天然病毒颗粒相似。经纯化的目的蛋白纯度接近100%。结论已成功构建了pPIC3·5K-HPV16L1/E7和pPIC9K-HPV16L1/E7重组表达载体,阳性转化菌株可表达结构与野生型HPV16一致的VLPs。

Cloning and Expression of L1/E7 Chimeric Gene of Human Papillomavirus Type 16 in Pichia pastoris

Objective To clone the L1/E7 chimeric gene of human papillomavirus type 16 and express in Pichia pastoris.Methods Amplify HPV16 L1/E7 gene by PCR and clone into expression vectors pPIC-3.5K and pPIC-9K to construct recombinant plasmids pPIC3.5K-HPV16 L1/E7 and pPIC9K-HPV16 L1/E7 respectively.Identify the recombinant plasmids by sequencing and transform to GS115 and KM71 strains of Pichia pastoris separately.Screen positive recombinants and identify by PCR,then express HPV16 L1/E7 chimeric protein in vitro under induction of methanol.Identify the expressed product by SDS-PAGE and Western blot.Observe the formation of virus-like particles(VLPs) by electron microscopy and purify by cesium chloride density gradient centrifugation.Results HPV16 L1/E7 gene was successfully inserted into vectors pPIC-3.5K and pPIC-9K.Six recombinant KM71 strains with HPV16 L1/E7-pPIC3.5K,five recombinant GS115 strains with HPV16 L1/E7-pPIC3.5K,one recombinant KM71 strain with HPV16 L1/E7-pPIC9K and two recombinant GS115 strains with HPV16 L1/E7-pPIC9K were obtained.The expressed product with a relative molecular weight of 69 000,which was consistent with that expected,contained 0.35-1.04 mg/ml protein.Western blot showed specific bindings of the expressed protein to the McAbs against HPV16 L1 and HPV16 E7 proteins.The VLPs with similar appearance to that of natural HPV16 particles were observed under electron microscope and reached a purity of nearly 100% after purification.Conclusion Recombinant plasmids pPIC3.5K-HPV16 L1/E7 and pPIC9K-HPV16 L1/E7 were successfully constructed and used for the expression of VLPs with identical structure to that of wild HPV16.