摘要
目的构建乙型肝炎病毒核心蛋白(HBc)突变体L60V重组质粒,并探讨L60V拮抗α干扰素(IFN-α)抗病毒活性的机制。方法构建HBc突变体融合表达载体pEGFPL60V,转染人肝胚瘤细胞株HepG2细胞,荧光显微镜及Western blot法检测其在细胞中的表达;并以RT-PCR及Western blot法检测JAK-STAT信号转导途径分子及抗病毒蛋白表达。结果经酶切和测序分析,成功构建HBc突变体L60V重组质粒;将L60V和HBc野生株表达质粒(pEGFP-WT)分别转染HepG2细胞后,以1 000 IU/ml IFN-α处理,转染L60V和HBc野生株表达质粒的细胞内抗黏液病毒A(MxA)及JAK-STAT信号转导途径分子STAT1、STAT2、IRF9表达减少,其中在转染pEGFP-L60V细胞内的MxA、STAT1、STAT2和IRF9表达显著减少。结论 HBc突变体L60V可能通过JAK-STAT信号转导途径抑制抗病毒蛋白MxA的表达来拮抗IFN-α的抗病毒作用;其拮抗IFN-α抗病毒作用比HBc更显著。
Objective To construct recombinant plasmid of L60V mutant core proteins of HBV, and explore its an-tagonistic action on the antiviral action of interferon-α（ IFN-α） . Methods Built the fusion expression vector pEG-FP-L60V of HBc mutant,the expression of the proteins was tested by the fluorescent microscope and Western blot after transfection to HepG2 cells. Detect moleculars of JAK-STAT signal transduction pathway and the antiviral pro-tein expression by RT-PCR and Western blot. Results The recombinant plasmid of L60V mutant core proteins of HBV was established successfully according to the result of restriction endonuclease digestion and DNA sequencing;HepG2 cells were transfected with pEGFP-L60V and pEGFP-WT, the expression of the antiviral proteins MxA and JAK-STAT signal transduction pathway moleculars were decreased after treatment with 1 000 IU/ml IFN-α, and de-creased even more in cells transfected with pEGFP-L60V. Conclusion L60V mutant core protein of HBV has the mechanism to antagonize the IFN-α antiviral activity by lowering antiviral protein MxA expression through JAK-STAT signal transduction pathway, and its functions are more efficiently than that of HBc.
出处
《安徽医科大学学报》
CAS
北大核心
2014年第2期181-185,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81171662)
安徽省卫生厅中医药计划科研基金(编号:2012ZY110)
