摘要
目的构建系列含乙型肝炎病毒(HBV)表面抗原前-前-S区的酵母细胞表达质粒,初步探讨表达蛋白是否具有反式激活作用。方法用多聚酶链反应(PCR)扩增含前-前-S区的全S、S2和S基因,定向克隆于pDEST32载体,进行序列测定,重组质粒命名为pDEST32-wS、pDEST32-pS2和pDEST32-SHBs。用乙酸锂转化法将重组质粒转化酵母菌MaV203,经Western blot和胶体金法验证重组质粒在酵母细胞中的表达。以酵母双杂交法将重组质粒和载体pDEST22共同转入酵母菌MaV203,并在SC/-leu/-trp/-his三缺培养基及不同浓度梯度3AT培养基中验证自激活。结果重组质粒pDEST32-wS经序列测定含有HBV自前-前-S至主蛋白基因序列。经Western blot和胶体金法证实转染的酵母细胞可表达表面抗原蛋白。pDEST32-wS与pDEST22共转染实验证实被转染酵母细胞不能在浓度30 mmol/L以上的3AT培养基生存。结论构建了pDEST32-wS、pDEST32-pS2和pDEST32-SHBs表达载体,pDEST32-wS和pDEST32-SHBs在酵母细胞中可表达HBsAg,含前-前-S区的HBV表面抗原可能不具有反式激活作用。
Objective To construct yeast expression vector of HBV whole-S gene including pre-pre-S region and explore the trans-activation function of whole-S protein.Methods Whole-S gene,pre-pre-S to pre-S2 region and major HBsAg coding region containing Not I and Sal I endoenzyme sites were obtained by PCR method.After enzyme digestion,PCR products were cloned into yeast expression vector pDEST32.Recombinant plasmids were sequenced and named as pDEST32-wS,pDEST32-pS2 and pDEST32-SHBs,respectively.Recombinant plasmids were transfected into yeast cell MaV203 by Liac-mediated transformation.Whole surface protein expressed in yeast cell was tested by Western blot and colloidal gold analysis.Recombinant plasmids pDEST32-wS,pDEST32-pS2 and pDEST32-SHBs were transfected into MaV203 by yeast two-hybrid and cultured in a series of 3AT plates with SC/-leu/-trp/-his to testify autonomous activation.Results After sequencing,recombinant plasmid pDEST32-wS was proved to include the anticipated fragment of whole S gene.Both Western blot and colloidal gold analysis showed that yeast cell transfected with plasmid pDEST32-wS can express whole-S protein.Yeast two-hybrid method showed that MaV203 transfected by pDEST32-wS and pDEST22 cannot grow in SC/-leu/-trp/-his plates with 3AT concentration higher than 30 mmol/L.Conclusions Recombinant plasmids pDEST32-wS,pDEST32-pS2 and pDEST32-SHBs were successfully constructed.MaV203 transfected by pDEST32-wS or pDEST32-SHBs can express HBsAg.HBV whole S protein might have no trans-activation ability.
出处
《中华实验和临床感染病杂志（电子版）》
CAS
2008年第3期146-152,共7页
Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)
基金
厦门市首批重大疾病科研攻关项目(WKZ0501)
厦门市卫生局医学科研立项项目(WSK0506)
厦门大学引进人才科研启动基金(Z03109)
福建省青年科技人才创新项目(2006F3127)
福建省青年科技人才创新项目(2006F3127)
作者简介
董菁 Email:dj@xmzsh.com 通讯作者
