真核表达载体pcDNA3.1(+)-vp28的构建及其在细胞中的表达  

Construction of Eukaryotic Expression Vector pcDNA3.1(+)-vp28 and Its Expression in Cell

作　　者：姜坤[1,2] 黄健[1,2] 张峥[2] 成君军[2,3] 石正丽[3] 刘庆慧[2] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]中国水产科学研究院黄海水产研究所,山东青岛266071 [3]中国科学院武汉病毒研究所,湖北武汉430071

出　　处：《安徽农业科学》2010年第22期11732-11734,共3页Journal of Anhui Agricultural Sciences

基　　金：国家重点基础研究发展计划课题(2006CB101801);国家高技术发展计划(863计划)课题(2006AA100312);公益性行业(农业)科研专项经费项目(200803012);国家虾现代产业技术体系(nycytx-46)共同资助

摘　　要：[目的]为研究对虾白斑综合征病毒(WSSV)囊膜蛋白与虾细胞之间的作用机制奠定基础。[方法]采用PCR扩增方法构建含白斑综合征病毒囊膜蛋白VP28的真核表达重组载体pcDNA3.1(+)-vp28,用磷酸钙共沉淀法将重组质粒转染到293T细胞中,采用SDS-PAGE和Western blot检测表达产物。[结果]VP28的PCR扩增产物在琼脂糖凝胶电泳中呈现出1条634bp左右的条带,与GenBank中vp28的序列完全一致。对pcDNA3.1(+)-vp28转染后的阳性单克隆菌落进行PCR鉴定、扩大培养和质粒提取,并对所得质粒进行HindⅢ和BamⅢ双酶切,获得了5409和610bp左右的2条带,与预期结果一致;所获扩增产物与vp28序列完全一致,说明重组质粒pcDNA3.1(+)-vp28被成功转染到293T细胞中;SDS-PAGE和Westernblot检测结果显示,pcDNA3.1(+)-vp28在293T细胞中的表达产物与预期结果相符。[结论]pcDNA3.1(+)-vp28在293T细胞中可成功表达VP28。[Objective] The aim was to lay a foundation for researching the action mechanism between peplos protein of white spot syndrome virus （WSSV） and cell in prawn.[Method] PCR amplification method was used to construct recombinant eukaryotic expression vector pcDNA3.1（＋）-vp28 of peplos protein v28 containing WSSV.The calcium phosphate co-precipitation method was used to transfect recombinant plasmid into 293T cell.The SDS-PAGE and Western blot were used to detect expression product.[Result] The PCR amplification products of VP28 showed a strip with length of about 634 bp in agarose gel electrophoresis and its sequence was completely identical with the sequence of vp28 in GenBank.The positive monoclone colonies transfected with pcDNA3.1 （＋）-vp28 were prepared for PCR identification,amplification culture and plasmid extraction;the obtained plasmids were prepared for HindⅢ and BamⅢ dual-enzyme digestion and 2 strips with lengths of about 5409 and 610 bp were obtained and this was identical with predicted results.The sequences of obtained amplification product and vp28 were identical completely,indicating that the recombinant plasmid pcDNA3.1（＋）-vp28 was transfected into 293T cell successfully.The results from SDS-PAGE and Western blot detections showed that the expression product of pcDNA3.1 （＋）-vp28 in 293T cell was identical with predicted results.[Conclusion] pcDNA3.1（＋）-vp28 could be expressed to be VP28 in 293T cell.

关 键 词：WSSV VP28 细胞转染 293T细胞 

分 类 号：R392.11[医药卫生—免疫学]