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目的:构建B19病毒XA株VPlu基因的真核表达载体和稳定转染细胞系,探讨B19病毒XA株VPl独特区蛋白对Hela细胞凋亡的影响。方法:采用本课题组前期构建的真核表达载体plRES2-EGFP-Vplu及plRES2-EGFP对照质粒,将其稳定转染至Hela细胞,通过流式细胞术检测EGFP阳性细胞的比例以确定稳定转染的细胞系是否成功构建;提取稳定转染的Hela细胞的总蛋白,通过Western blot检测细胞内凋亡相关基因Caspase3的表达;转染plRES2-EGFP-Vplu及plRES2-EGFP的Hela细胞经过Annexin V和PI的染色后,通过流式细胞术检测其凋亡率。结果:本实验成功构建了plRES2-EGFP-VPlu稳定转染的Hela细胞系,plRES2-EGFP-Vplu稳定转染的Hela细胞中Caspase3的表达较转染对照质粒的Hela细胞明显增加,细胞凋亡率亦显著升高,差异均具有统计学意义(P<0.05)。结论:B19病毒XA株VP1独特区蛋白能够显著促进Hela细胞的凋亡。
OBJECTIVE: To construct eukaryotic expression vector and stable transfected cell line of VP19 gene of B19 virus XA strain and study the effect of VP1 unique domain protein of B19 virus XA strain on Hela cell apoptosis. Methods: The eukaryotic expression vectors plRES2-EGFP-Vplu and plRES2-EGFP constructed in our study were stably transfected into Hela cells. The proportion of EGFP positive cells was determined by flow cytometry to confirm the stable transfection Of the transfected Hela cells were transfected with plRES2-EGFP-Vplu and plRES2-EGFP by Annexin V and Annexin V, and the expression of Caspase3 was detected by Western blot. After PI staining, the apoptosis rate was detected by flow cytometry. Results: The HeLa cell line stably transfected with plRES2-EGFP-VPlu was successfully constructed. The expression of Caspase3 in HeLa cells stably transfected with plRES2-EGFP-Vplu was significantly higher than that of Hela cells transfected with control plasmid. The apoptosis rate Also significantly increased, the differences were statistically significant (P <0.05). Conclusion: The VP1 unique region of B19 virus XA strain can significantly promote the apoptosis of Hela cells.