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.
出处
《中华微生物学和免疫学杂志》
CSCD
北大核心
2000年第5期477-480,共4页
Chinese Journal of Microbiology and Immunology
