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目的本研究拟探讨HCV非结构蛋白5A(NS5A)对HIV长末端重复序列(long terminal repeat,LTR)影响,从而为HCV对HIV的影响提供实验依据。方法将构建的LTR启动子驱动的荧光素酶(Luc)报告基因表达质粒(pGL3-LTR-Luc)和含HCV NS5A基因的表达质粒pCNS5A共转染肝癌细胞株(Huh7细胞),采用免疫细胞化学技术、Western Blot及逆转录聚合酶链反应检测HCV NS5A蛋白及mRNA的表达;本实验分三组,将质粒pGL3-LTR-Luc(空白组)、质粒pRc/CMV+pGL3-LTR-Luc(对照组)、质粒pCNS5A+pGL3-LTR-Luc(实验组)分别转染Huh7细胞,48 h后收集细胞,采用Luc活性检测LTR的活性,以观察HCV NS5A对LTR的调控影响。所得荧光活性值以均数±标准差表示,采用Levene’s方差齐性检验,多组间比较采用单因素方差分析,两两比较行LSD-t检验。结果转染pcNS5A质粒的Huh7细胞质经RT-PCR及Western Blot检测,HCV NS5A mRNA及蛋白在细胞中获得表达。方差分析结果提示LTR荧光活性在三组间有明显的差异(F=7.876,P=0.002),进一步比较各组间的差异,结果提示共转染质粒pcNS5A+pGL3-LTR-Luc组的Huh7细胞中Luc相对活性(22 476±4471)明显高于单转染pGL3-LTR-Luc组(15 887±3039,P=0.002)及共转染质粒pRc/CMV+pGL3-LTR-Luc组(16 321±4162,P=0.008),差异有统计学意义。结论表达HCV NS5A的质粒pCNS5A成功转染至Huh7细胞;HCV NS5A蛋白能激活HIV LTR,提示HCV NS5A可能为HCV促进HIV复制的分子机制之一。
Objective This study was designed to investigate the effect of HCV nonstructural protein 5A (NS5A) on long terminal repeat (LTR) in HIV and to provide experimental evidence for the influence of HCV on HIV. Methods The luciferase (Luc) reporter gene expression plasmid (pGL3-LTR-Luc) and the expression vector pCNS5A containing HCV NS5A gene were co-transfected into Huh7 cells (Huh7 cells). The immunocytochemistry Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of HCV NS5A protein and mRNA. Plasmids pGL3-LTR-Luc (blank group), plasmid pRc / CMV + pGL3-LTR- Huh7 cells were transfected with plasmids pCNS5A + pGL3-LTR-Luc (experimental group), and the cells were harvested 48 h later. The activity of LTR was detected by Luc activity in order to observe the effect of HCV NS5A on LTR. The fluorescence activity values were expressed as mean ± standard deviation, Levene’s test of homogeneity of variance, one-way ANOVA between groups and LSD-t test. Results The Huh7 cytoplasm of pcNS5A plasmid was detected by RT-PCR and Western Blot. HCV NS5A mRNA and protein were expressed in the cells. Variance analysis suggested that there was a significant difference in LTR fluorescence activity between the three groups (F = 7.876, P = 0.002). The differences between the groups were further compared. The results showed that Huh7 cells co-transfected with plasmid pcNS5A + pGL3-LTR-Luc The relative activity of Luc in 22 476 ± 4471 cells was significantly higher than those in pGL3-LTR-Luc transfected cells (15 887 ± 3039, P = 0.002) and co-transfected plasmid pRc / CMV + pGL3- ± 4162, P = 0.008), the difference was statistically significant. Conclusion HCV NS5A plasmid pCNS5A was transfected into Huh7 cells successfully. HCV NS5A protein could activate HIV LTR, suggesting that HCV NS5A may be one of the molecular mechanisms by which HCV promotes HIV replication.