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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、研制登革病毒诊断试剂及核酸疫苗奠定了基础
Objective To construct eukaryotic expression vector of dengue virus type 2 E gene and realize the eukaryotic expression of dengue virus E protein. Methods The full length of envelope glycoprotein E gene of dengue virus type 2 (NGC strain) was amplified by reverse transcription polymerase chain reaction (RT PCR) and cloned into the downstream of Pcmv promoter of eukaryotic expression vector pcDNA3 to construct recombinant eukaryotic expression vector The plasmid pcDNA3 E was transfected into NIH3T3 cells by lipofection. The expressed products were analyzed by immunofluorescence, SDS PAGE and Western blotting. Results Recombinant eukaryotic expression plasmid pcDNA3 E was successfully constructed and transfected into NIH3T3 cells by lipofection. Immunofluorescence, SDS PAGE and Western blotting analysis showed that E gene was expressed in NIH3T3 cells. The relative molecular mass (Mr) is 60 × 10 3. Conclusion The construction of eukaryotic expression vector of dengue virus E gene and eukaryotic expression of E gene laid the foundation for the study of the structure and function of dengue virus E protein and development of dengue virus diagnostic reagent and nucleic acid vaccine