目的:构建丙型肝炎病毒核心蛋白(HCV-core-1b)真核重组质粒,获得稳定表达HCV-core-1b的HepG2细胞株,观察HCV-core-1b对HepG2细胞株生长周期及cyclin D1 和pRb/p130表达的影响,探讨丙型肝炎病毒慢性感染的可能机制.方法:将HCV-core-1b亚克隆入pBabe-Flag-puro载体,获得重组质粒pBabe-Flag-HCV-core-1b;将重组质粒转染病毒包装细胞Pheonix 293T,筛选获得分泌HCV-core-1b的病毒包装细胞株.利用包装细胞产生的病毒上清感染靶细胞,筛选后获得稳定表达HCV-core-1b的HepG2细胞株,流式细胞仪检测靶细胞生长周期的变化,Western blotting检测cyclin D1 和pRb/p130蛋白的表达.结果:基因测序确认HCV-core-1b亚型基因编码区完整无移位,与标签蛋白Flag形成融合蛋白.HepG2-HCV-core细胞株成功表达Flag-HCV-core-1b蛋白,并导致细胞cyclin D1 和 pRb/p130的水平下调,显著改变了HepG2细胞生长周期,使细胞阻滞在G0/G1期.结论:成功构建了pBabe-Flag-HCV-core-1b真核表达质粒,获得稳定表达Flag-HCV-core-1b融合蛋白的HepG2细胞.由于HCV-core-1b蛋白的表达,下调了HepG2细胞 cyclin D1和pRb/p130的表达,显著抑制HepG2细胞生长周期.%AIM: To investigate the molecular mechanism of hepatitis C virus ( HCV ) chronic infection by studying the effect of its core protein on cell growth and the expression of cell cycle regulators such as cyclin D 1 and pRb/ pl30 in HepG2 cells. METHODS: A eukaryotic expression vector that carried a gene encoding HCV - core - 1b was constructed. The cDNA of HCV core protein was subcloned into pBabe - Flag - puro vector to generate pBabe - Flag - HCV -core - lb. The plasmid was transfected into Pheonix 293T packaging cells to produce retroviruses. The virus - containing supernatant collected from the cell culture was used to infect HepG 2 cells and subsequently the cell line that stably ex -pressed the core protein of HCV was obtained. The cell cycle was analyzed by flow cytometry. The expression of cyclin D1 and pRb/pl30 was examined by Western blotting. RESULTS; The pBabe - Flag - HCV - core - lb vector was confirmed by DNA sequencing. The expression of HCV gene type 1b core protein was verified by Western blotting. The overexpression of HCV gene type lb core protein impaired the cell cycle progression in G0/G1 phase and significantly reduced the levels of cyclin Dl and pRb/pl30 in the cells. CONCLUSION; A eukaryotic expression plasmid that contains the cDNA of HCV core protein is successfully constructed , and a HepG2 cell line which stably expressed the core protein of HCV is also es -tablished. HCV gene type lb core protein inhibits the cell cycle possibly through down - regulation of cyclin Dl and pRb/ pl30 proteins in the cells.
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