四环素诱导的丙型肝炎病毒非结构蛋白5B稳定细胞株的构建和检测

Establishment and detection of a tetracycline-inducible stable cell line expressing hepatitis C virus nonstructural protein 5B

丙型肝炎病毒(HCV)感染个体后在宿主细胞内长时间保持低水平复制,与慢性肝炎、肝硬化及肝细胞肝癌的发生密切相关。目前,HCV感染后肝细胞发生转化的具体机制还不清楚。非结构蛋白5B(NS5B)是HCV编码的非结构蛋白之一,具有RNA依赖的RNA聚合酶活性(RdRp),是病毒复制所需的关键酶。除参与病毒复制外,NS5B通过直接与宿主蛋白相互作用影响细胞的正常功能逐渐成为人们关注的焦点。为深入了解NS5B在病毒复制和致病过程中的作用,本研究在肝癌细胞系HepG2细胞中构建了可由四环素诱导(Tet-On)的NS5B稳定细胞株。结果显示,与对照组相比,加入四环素可成功诱导NS5B。加入不同浓度的四环素或经过不同的诱导时间,目的蛋白均能同时被标签抗体和抗NS5B抗体检测。免疫荧光法进一步证实NS5B存在于细胞质内。本研究所构建的NS5B稳定细胞株可用于诸多后续试验,为深入研究NS5B的功能提供了细胞模型。

Hepatitis C virus（HCV） infection is a major risk factor of chronic hepatitis,liver cirrhosis,and hepatocellular carcinoma（HCC）.However,the detailed mechanism of action on cancer caused by HCV is still elusive.Nonstructural protein 5B（NS5B） is one of the nonstructural proteins encoded by HCV and is essential for HCV RNA replication due to its RNA-dependent RNA polymerase（RdRp） activity.In addition to participating in viral RNA replication,NS5B was also found to be involved in the pathogenicity of HCV through a direct interaction with host proteins.In order to address the role of NS5B in HCV replication and pathogenicity,we constructed a tetracycline-inducible（Tet-On） stable cell line expressing NS5B in HepG2 cells.Protein expression was examined by Western blot using both tag antibody and anti-NS5B antibody induced by doxycycline either at different concentrations or at different indicated time points.Moreover,immunofluorescence assay further confirmed the results.In conclusion,the HepG2 Tet-On NS5B stable cell line may be a useful model to investigate the role of NS5B in HCV replication and pathogenicity.