在四环素调控系统下表达HA蛋白MDCK细胞系的建立

Establishment of MDCK Cell Lines Expressing HA Protein under Tetracycline Regulation System

细胞培养流感病毒生产疫苗过程中细胞系的选择至关重要,本研究建立了可控表达H9N2亚型禽流感病毒(AIV)HA蛋白的MDCK细胞系,能有效支持H9N2流感病毒的扩增。本研究应用插入四环素调控元件和H9N2亚型禽流感病毒HA基因的重组质粒V2-H9,与辅助质粒pMDSV、psPAX2共转染293T细胞包装慢病毒,将包装出的慢病毒感染MDCK细胞,通过添加嘌呤霉素筛选出阳性的单克隆细胞。再用PR8 SH441病毒感染筛选出来的MDCK单克隆细胞。结果显示,流感病毒在其中4株MDCK单克隆细胞的HA效价接近于8;生长曲线结果显示,流感病毒在第34株MDCK单克隆细胞中复制效果最好;间接免疫荧光(IFA)和Western blot检验结果显示,第34株MDCK单克隆细胞H9 HA的表达量最高;将第34株MDCK单克隆细胞驯化后进行悬浮培养,每隔24 h测定细胞密度,结果表明,第34株MDCK单克隆细胞可以悬浮培养,且流感病毒在第34株MDCK悬浮细胞中HA效价可达9log2。以上结果表明,本研究获得了一株可高效扩增H9亚型禽流感病毒的MDCK细胞株,为流感弱毒疫苗的研发提供了新的研究方法和实验基础。

The choice of cell line in the process of cell culture virus production vaccine is very important.In this study,an MDCK cell line that can controllably express the H9N2 subtype avian infl uenza virus(AIV)HA protein was established.This cell line is expected to be used as a cell for the production of vaccines from the amplifi ed infl uenza virus.In this study,the recombinant plasmid V2-H9 inserted into the tetracycline regulatory element and the HA gene of the H9N2 subtype avian infl uenza virus was used to co-transfect 293T cells with the helper plasmids pMDSV and psPAX2 to package the lentivirus.The packaged lentivirus was infected with MDCK cells,the positive monoclonal cells were screened by puromycin.Through hemagglutination test,it was found that the HA titer of 4 strains of MDCK monoclonal cells of infl uenza virus was close to 8. Through the virus growth curve, it was found that the infl uenza virus replicated best in the 34th MDCK monoclonal cell. Indirect immunofl uorescence (IFA) and western blot were used to identify the expression of H9 HA in the 34th MDCK monoclonal cell. The 34th MDCK monoclonal cells were acclimatized and subjected to suspension culture, and the cell density was measured every 24 h. The results showed that the 34th MDCK monoclonal cells could be cultured in suspension. The hemagglutination test proved that the HA titer of infl uenza virus in the 34th MDCK suspension cell can reach 9log2.