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目的在前期研究结果提示Glt25D2与HBV包膜蛋白大蛋白(LHBs)在体外相互作用基础上证明Glt25D2是否与HBV LHBs分泌相关。方法采用激光共聚焦方法分析Glt25D2与LHBs在HepG2细胞内的定位。免疫共沉淀方法进一步证实Glt25D2与LHBs的相互作用。采用实时荧光定量PCR和Western blot方法分析mRNA和蛋白表达水平。应用ELISA方法检测细胞上清LHBs水平。实时荧光定量PCR方法检测上清HBV病毒载量。ELISA方法检测上清HBV LHBs水平。Western blot方法检测细胞中LHBs蛋白含量。Cobas Amplicor HBV Monitor Test方法检测细胞上清液HBV DNA载量。结果 Glt25D2与LHBs在体外相互作用,上调的Glt25D2高表达促进HBV DNA复制和LHBs表达,Glt25D2低表达抑制HBV DNA复制和LHBs表达。结论 Glt25D2与乙型肝炎包膜蛋白大蛋白的分泌有关。
Objective The results of previous studies suggest that Glt25D2 and HBV envelope protein large protein (LHBs) on the basis of the interaction in vitro to prove whether Glt25D2 HBV LHBs secretion. Methods The localization of Glt25D2 and LHBs in HepG2 cells was analyzed by laser scanning confocal microscopy. Immunoprecipitation method further confirmed the interaction of Glt25D2 and LHBs. The mRNA and protein expression levels were analyzed by real-time fluorescence quantitative PCR and Western blot. ELISA method was used to detect the level of LHBs in the supernatant. Real-time fluorescence quantitative PCR method was used to detect the supernatant HBV viral load. ELISA method to detect the level of HBV LHBs in supernatant. Western blot method to detect the content of LHBs protein in cells. The Cobas Amplicor HBV Monitor Test method was used to detect the HBV DNA load of the cell supernatant. Results Glutathione dehydrogenase (Glt25D2) interacted with LHBs in vitro. High expression of Glt25D2 increased HBV DNA replication and LHBs expression, while low Glt25D2 expression inhibited HBV DNA replication and LHBs expression. Conclusion Glt25D2 is associated with the secretion of hepatitis B envelope protein.