包装西尼罗假病毒稳定细胞株的建立

Construction of a stable transgenic cell strain for packaging of West Nile pseudovirus

目的 为方便制备西尼罗假病毒,建立可包装西尼罗假病毒的稳定细胞系。方法 构建表达西尼罗病毒(WNV)结构蛋白C、prM和E蛋白的重组质粒pcDNA3.1-CME,转染BHK21细胞后,G418(1 mg/ml)加压筛选;利用流式细胞术、间接免疫荧光染色(IFA)以及Western印迹确证稳定细胞株中3种结构蛋白C、prM以及E蛋白的表达;利用103稀释的含海肾荧光素酶报告基因的WNV假病毒上清感染稳定细胞株,检测荧光素酶活性。结果与结论 所构建的稳定细胞株表达结构蛋白C、prM以及E蛋白;其被WNV假病毒感染后分泌的上清进一步感染BHK21细胞后,BHK21细胞产生荧光,证实该稳定细胞株能够包装出WNV假病毒。

Objective To establish a stable cell strain that can package West Nile pseudovirus in order to facilitate the preparation of West Nile pseudovirus. Methods BHK21 cells were transfected with recombinant plasmid pcDNA3.1-CME expressing the structural proteins C,prM and E of West Nile virus(WNV)before endergoing pressured screening with G418(1 mg/ml). The three structural proteins C,prM and E expressed in stable cell strains was identified by flow cytometry,indirect immunofluorescence staining(IFA)and Western blotting(WB). The stable cell strain were infected with the 103-diluted WNV pseudovirus supernatant containing the luciferase reporter gene,and the infected cells were lysed to detect the luciferase activity. Results and Conclusion The stable cell strain expressed three structural proteins:C,prM and E.The supernatant secreted by the WNV pseudovirus was further infected with BHK21 cells that were to produce fluorescence,confirming that the stable cell strain could package the WNV pseudovirus.