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...
【英文摘要】 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...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
