乙型肝炎病毒核心蛋白原蛋白转化酶切割假定产物的体外研究

目的 通过体外试验探讨furin切割核心蛋白的可能性,并通过重组载体表达,研究其假定产物在HepG2细胞内的分布特点. 方法 重组HBV核心蛋白,并将其在不同温度和时间条件下(4℃消化16 h、30℃消化1h和30℃消化3h)接受furin切割,产物采用Western blot分析.构建HBV核心蛋白furin切割假定产物的真核表达载体,并以完整核心蛋白表达载体作为对照,转染HepG2细胞,获得稳定表达细胞株.使用核心蛋白与假定产物共同区的单克隆抗体进行间接免疫荧光染色,用共聚焦显微镜观察其细胞内分布. 结果 HBV核心蛋白在多种条件下均被furin切断,其主要产物相对分子质量约为15 000,与预期相符,且30℃切割1 h的效果最好.真核表达的核心抗原假定切割产物能与核心蛋白单克隆抗体结合,且在细胞内的分布与完整核心蛋白类似,主要以颗粒形式分布在细胞质. 结论 HBV核心蛋白在体外能被furin切割,其主要产物与完整核心蛋白有类似的免疫原性和相同的细胞内分布,提示furin或其家族成员参与了HBV复制调节,其切割产物对机体抗病毒免疫可能存在深远影响,值得深入研究.

Study on a putative, proprotein convertase-cleaved product of HBV core protein in vitro

Objective To investigate the cleavage of HBV core protein in vivo by proprotein convertase furin or its family members and observe the intracellular localization of the putative cleaved product.Methods Recombinant HBV core protein was incubated with furin under different conditions in vitro, and the reaction was checked with Western blotting. The recombinant vectors expressed the putative cleaved fragment and intact core protein (serves as control) were constructed. The stable expression cell lines were established by transfecting constructs into HepG2 cell line, for which indirect immunofluorescence staining was used by monoclonal anti-HBc against the region shared by core protein and its cleaved product .The confocal microscopy was carried out to observe the intracellular distribution. Results HBV core protein was cleaved by furin in vitro under different tested conditions. The molecular weight of the major cleaved product just about 15 000 was in concordance with the expectation. The expressed cleaved fragment could react to the monoclonal antibody against core protein, and mainly located in cytosol in particle style just like the intact core protein. Conclusion HBV core protein can be cleaved by furin in vitro. The major cleaved product has similar antigenicity and subcellular distribution to core protein. These data suggest that proprotein convertase furin or its family members play important roles in HBV replication regulation, and the cleaved product may be involved in antiviral immunity of HBV infection. Further investigations are imperative.