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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 the anti-α-interferon (IFN-α) -related hepatocyte proteins that bind to the N-terminal 257 amino acids of hepatitis B virus DNA polymerase (TP257) and to explore the mechanism of TP257 anti-IFN-α. Methods TP257 adenovirus shuttle vector pShuttle-IRES-hrGFP-TP257 was constructed and recombined with the adenovirus backbone vector pADEasy-1. The recombinant adenovirus was packaged with 293A cells. Huh7 cells were infected with recombinant adenovirus and induced by IFN-α, cell proteins were harvested, and the expression of TP257 protein in the cells was confirmed by Western blot. Cell proteins were immunoprecipitated and differentially expressed by mass spectrometry after SDS-PAGE. Results The constructed recombinant adenovirus AD-TP257 effectively infected Huh7 cells and overexpressed TP257 protein. Huh7 cells were treated with IFN-α after infection with AD-TP257, and four kinds of differential proteins were screened by immunoprecipitation. Three of them were identified by peptide mass fingerprinting as heat shock protein 60, histone HIST1H2BC and myosin regulatory light chain 12A. Conclusion The anti-IFN-α effect of TP257 may be related to its interaction with specific hepatocyte proteins.