乙型肝炎病毒X蛋白升高细胞内钙离子机制研究

The Mechanism of Hepatitis B Virus X Protein Elevating Cytosolic Calcium Signals

目的研究乙型肝炎病毒X蛋白(HBx)调节细胞内钙离子可能的分子机制,揭示乙型肝炎病毒(HBV)诱导肝癌的可能途径。方法培养人胚肾细胞(HEK293),取第2代HEK293细胞共转染HBx基因、钙释放激活钙通道蛋白1(Orai1)基因或者间质相互作用因子1(STIM1)基因,免疫共沉淀(co-IP)实验观察细胞内蛋白结合情况,并使用谷胱甘肽巯基转移酶(GST)pull-down实验研究HBx与Orai1作用位点,使用钙离子测量/成像系统检测HBx蛋白对细胞内钙离子的影响。结果转染后细胞生长状态良好,co-IP实验结果显示HBx蛋白在细胞内可以与Orai1蛋白结合,GST pull-down实验显示HBx蛋白可以与Orai1蛋白C末端结合,钙离子测量/成像系统检测显示HBx蛋白可升高活细胞钙内流。结论 HBx蛋白通过与细胞膜钙离子通道Oria1结合蛋白的C末端结合,增加细胞钙内流,扰乱细胞内钙离子平衡,从而影响细胞增殖等活性。

Objective To examine the possible mechanism by which HBx protein regulates the intracetlular calcium lev- els. Methods Plasmids pcDNA-Flag-HBx,pcDNA-HA-Orail ,pcDNA HA-STIM1 and pEGFP-C3-HBx were co-transfected in- to HEK293 cells. The interaction between SOC components and HBx protein was confirmed in co-immunoprecipitation（Co-IP）, and the interaction of Orail protein with HBx protein was assessed by using Glutathione S transferase （GST）pull-down assays. Single-cell calcium measurements were made to determine the effect of HBx protein on intracellular calcium lev- els. Results The transfected cells grew well. Co-IP assay revealed that HBx protein could bind to intracellular Orail pro- tein. GST pull-down assay showed that HBx protein interacted with the C-terminus of Orail. Subsequent single-cell calcium measurements demonstrated that HBx protein could increase the calcium influx of viable cells. Conclusion HBx protein regu- lates SOCE via direct interaction with the C-terminus of Orail protein, and HBx protein may elevate the intracellular calcium levels,disturb the intracellular calcium balance,thereby influencing the proliferation and other activities of cells.