登革2型病毒NGC株E基因在NIH3T3细胞中的表达及初步鉴定  

The expression of E gene region of Dengue 2 virus in NIH3T3 and its identification

作　　者：洪帮兴[1] 江丽芳[2] 张欣[2] 曹艳英[3] 郭辉玉[2] 张世英 

机构地区：[1]中山医科大学 [2]中山医科大学微生物学教研室 [3]暨南大学组织和器官移植研究中心 [4]深圳市福田卫生防疫站,518033

出　　处：《中华微生物学和免疫学杂志》2000年第5期477-480,共4页Chinese Journal of Microbiology and Immunology

摘　　要：目的　构建登革 2型病毒E基因的真核表达载体 ,实现登革病毒E蛋白的真核表达。方法　采用逆转录 多聚酶链反应 (RT PCR)扩增登革 2型病毒 (NGC株 )包膜糖蛋白E基因全长片段 ,克隆入真核表达载体pcDNA3的Pcmv启动子下游 ,构建重组真核表达质粒pcDNA3 E ,用脂质体转染法转染NIH3T3细胞 ,表达产物以免疫荧光、SDS PAGE和蛋白质印迹进行分析检测。结果　成功构建了重组真核表达质粒pcDNA3 E ,通过脂质体转染法导入NIH3T3细胞 ,免疫荧光、SDS PAGE和蛋白质印迹分析检测表明 ,E基因在NIH3T3细胞实现了真核表达 ,产物相对分子质量 (Mr)为 6 0× 10 3。结论登革病毒E基因真核表达载体的构建及E基因的真核表达为研究登革病毒E蛋白的结构与功能。Objective Construction of an eukaryotic expression plasmid of E gene fragment from Dengue 2 virus and eukaryotic expression of E glycosylated protein. Methods The E gene fragment of Dengue 2 virus NGC strain was amplified by RT PCR and this fragment was cloned into eukaryotic expression vector pcDNA3. The recombinant plasmid pcDNA3 E was thus constructed and identified by sequencing and digestion with restriction enzymes. The recombinant plasmid pcDNA3 E was transfected into NIH3T3 cell by lipofectin. The expressed protein was analyzed by immunofluorescence, SDS PAGE and Western blotting assay. Results A recombinant eukaryotic expression plasmid pcDNA3 E was successfully constructed. The recombinant plasmid expressed a 60×10 3 protein of Dengue 2 virus. Conclusions The constructions of eukaryotic expression of E gene fragment from Dengue 2 virus and eukaryotic expression of E glycosylated protein will be helpful to study the structure and functions of the E protein, and to the development of Dengue diagnostic agents as well as Dengue virus nucleic acid vaccine.

关 键 词：E基因 载体 真核表达 NIH3T3细胞 登革2型病毒 

分 类 号：R373[医药卫生—病原生物学]