乙型肝炎病毒L60V、I97L变异核壳蛋白对HepG2细胞HLA-A表达的影响

Effects of L60V and I97L point mutation in hepatitis B core protein on the expression of HLA-A molecule in HepG2 cells

目的研究L60V、I97L变异核壳蛋白对HepG2细胞HLA-A表达的影响。方法构建EGFP-野毒株核壳蛋白、L60V、I97L变异核壳蛋白表达载体,酶切和测序鉴定,克隆扩增后转染HepG2细胞,筛选阳性细胞株;荧光显微镜观察荧光蛋白的表达,Western blotting检测核壳蛋白的表达;RT-PCR检测HLA-A mRNA的表达,流式细胞术观察HLA-A蛋白的表达。结果成功建立了融合表达蛋白表达载体;荧光显微镜显示细胞内融合蛋白表达,Western blotting检测各细胞系蛋白表达的量基本相同;RT-PCR和流式细胞术结果表明,与野毒株相比I97L变异核壳蛋白表达细胞株HLA-A mRNA和蛋白表达均上调(P<0.05),而L60V变异核壳蛋白表达细胞株则均明显下调(P<0.05)。结论L60V、I97L变异核壳蛋白对HepG2细胞HLA-A表达与野毒株核壳蛋白的影响不尽相同,其在功能上与野毒株核壳蛋白存在差异。

To explore the effects of L60V and 197L point mutation in hepatitis B core protein on the HLA-A expression in HepG2 cells, the expression vectors for various mutant HBV core proteins and EGFP-wide type core protein were constructed, subsequently enzyme-digested and sequenced. These vectors were then transfected to HepG2 cells and the positive clone cells were screened by neomycin. The expression of fusion proteins was detected by fluorescent microscopical examination and measured by Western blotting, while the expressions of HLA-A mRNA and protein were tested with RT-PCR and flow cytometry assay respectively. The experimental results showed that the expression vectors for various fusion proteins （pEGFP-WT, pEGFP-V60 and pEGFP-L97） were successfully established and the amounts of the protein expression differed very little among various cell clones. However, as demonstrated by RT-PCR and flow cytometry assay the expressions of HLA-A mRNA and protein were up-regulated in pEGFP-L97 cell clone, but they were down-regulated in pEGFP-V60 cell clones in comparison with that of pEGFP-WT cell clones. It is concluded that the influence of L60V or 197L mutant hepatitis B core protein on the HLA-A expression in HepG2 cells varies a great deal in comparison with that of the wlde-type and this may be the possible reason by which they are hardly detected in patients with chronic active hepatititis B.