
丙型肝炎病毒核心基因置换对病毒复制和感染影响的初步研究
Preliminary study on influnce of core gene substitution in replication and infection of hepatitis C virus
本文旨在探讨丙型肝炎病毒(HCV)核心(core)蛋白在病毒复制和感染中的作用,采取基因置换的方法,用HCV 1b型J4株的核心基因平行置换2a 型J6JFH1株的核心区,构建了FL-J6JFH/J4core嵌合复制子。体外制备RNA转录体,以脂质体介导转染Huh7.5.1肝癌细胞。转染后第5天,荧光定量PCR(FQ-PCR)方法检测细胞内的HCV RNA水平。第8天,以丙型肝炎患者血清为一抗,免疫荧光染色法(IFA)检测转染细胞内HCV蛋白表达。同时收集转染后第8天的细胞上清液,感染naïve Huh7.5.1肝癌细胞,72小时后 IFA检测HCV蛋白表达。结果显示,FL-J6JFH/J4core 转染组第5天细胞内RNA水平与野生型FL-J6JFH1接近,无显著差异(n=4,P>0.05)。免疫荧光检测发现,FL-J6JFH/J4core 转染细胞后第8天及其上清液感染naïve Huh7.5.1细胞的HCV阳性细胞数都低于野生型。本研究结果初步表明,HCV各株间核心基因的替换会影响HCV的蛋白翻译和感染性病毒颗粒的产生与释放。
In order to explore the role of core protein on the replication and infection of hepatitis C virus (HCV), intergenotypic chimeric FL-J6JFH/J4 core replicon was constructed in which the core gene of Fl-J6JFH strain (genotype 2a) was replaced by corresponding gene from HC-J4 strain (1b). In vitro RNA transcripts of FL-J6JFH and FL-J6JFH/J4 core were prepared and transfected into Huh7.5.1 hepatocytes respectively with liposomes. At day 5 post-transfection, total cellular RNA was isolated and determined by fluorescence quantitative polymerase chain reaction (FQ-PCR) method. Immunofluorescence staining analysis was performed to test the expression of HCV proteins in transfected cells at day 8 post-transfection. The naïve Huh7.5.1 cells were inoculated with the supernatants collected at day 8 post-transfection, and HCV proteins were detected by immunofluorescence at 72 h after inoculation. The results demonstrated that, the intracellular HCV RNA values of FL-J6JFH/J4 core was close to that of wild-type FL-J6JFH1 at day 5 post-transfection, and no significant differences were observed (n=4, P>0.05). Immunofluorecene analysis showed that, as compared with that in the wild type cells, the expression of HCV proteins in either cells transfected with FL-J6JFH/J4 core RNA at day 8 post-transfection or inoculation with supernatants collected at day 8 post-transfection was significantly lower. The results suggest that the core gene substitution among different HCV strains might influence the HCV protein expression and the production of infectious viral particles.
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