乙型肝炎病毒DNA聚合酶TP257抗α-干扰素机制初步探讨

Preliminary investigation of the anti-IFN-α effect of the TP257 protein of hepatitis B virus DNA polymerase

目的筛选与乙型肝炎病毒DNA聚合酶N端257个氨基酸(TP257)相结合的抗α-干扰素(IFN-α)相关性肝细胞蛋白,初步探讨TP257抗IFN-α作用机制。方法构建TP257腺病毒穿梭载体pShuttle-IRES-hrGFP-TP257,并与腺病毒骨架载体pADEasy-1重组,以293A细胞包装重组腺病毒。用重组腺病毒感染Huh7细胞并以IFN-α诱导,收获细胞蛋白,Western blot验证TP257蛋白在细胞中的表达。细胞蛋白进行免疫沉淀,SDS-PAGE分离后通过质谱鉴定差异蛋白。结果构建的重组腺病毒AD-TP257能有效感染Huh7细胞并大量表达TP257蛋白。Huh7细胞感染AD-TP257后以IFN-α处理,并通过免疫沉淀筛选到4种差异蛋白,其中3种经肽质量指纹图谱鉴定分别为热休克蛋白60、组蛋白HIST1H2BC和肌凝蛋白调节轻链12A。结论 TP257抗IFN-α效应可能与它和特定肝细胞蛋白结合并相互作用有关。

Objective To screen for the cellular proteins that combine with the TP257 protein of HBV DNA polymerase and preliminarily investigate the mechanism of the anti-IFN-α effects of TP257. Methods The adenovirus shuttle vector pShuttle-IRES-hrGFP-TP257 was constructed and rccombined with adenovirus backbone vector pADEasy-1. The recombinant adenovirus particles were packaged and harvested in 293A cells. Huh7 cells were infected with recombinant adenoviral particles and treated with IFN-α . Expression of TP257 was verified by Western blotting, and cell proteins were subjected to immunoprecipitation. After separation by SDS-PAGE, the differentially precipitated proteins were identified by mass spectrometry. Results Recombinant adenovirus particles AD-TP257 were successfully generated and effectively infected Huh7 cells and also expressed the TP257 protein. After treatment of AD-TP257-infected Huh7 cells with IFN-α, four proteins were immunoprecipitated. Three were identified by MALDI-TOF-MS as a 60 ku heat shock protein, HIST1H2BC protein, and myosin regulatory light chain 12A, respectively. Conclusion Anti-IFN-α effects of TP257 may result from its binding to and interaction with certain cellular proteins.