丙型肝炎病毒核心蛋白稳定表达肝癌细胞株的构建及生物学研究  

Development of a Hepatoma Stable Cell Line Expressing HCV Core Protein

作　　者：单晓亮[1] 刘娇[1] 陈庆美[1] 周帆[1] 陈林林[1] 权会琴[1] 唐霓[1] 

机构地区：[1]重庆医科大学附属第二医院病毒性肝炎研究所感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《生物技术通报》2012年第1期99-103,共5页Biotechnology Bulletin

基　　金：国家自然科学基金资助项目(30972586);重庆市自然科学基金重点项目(CSTC;2009BA5036)

摘　　要：旨在构建稳定表达HCV核心蛋白的稳定细胞系Huh7-Core并进行初步的生物学功能研究。利用PCR技术扩增HCV核心蛋白基因,通过酶切连接反应将目的基因克隆至载体pSEB-3Flag中,将重组质粒pSEB-3F-Core和辅助质粒pAmpho共转染Huh7细胞,经过Blasticidine抗性筛选,建立稳定表达HCV核心蛋白的肝癌细胞系Huh7-Core。采用RT-PCR、Western blot鉴定Huh7-Core细胞株中核心蛋白的稳定表达并采用MTS、结晶紫试验观察Huh7-Core稳定细胞株的增殖情况。结果显示,成功构建了表达HCV核心蛋白的稳定细胞株Huh7-Core。结晶紫、MTS试验证实Huh7-Core细胞较Huh7-3Flag细胞增殖速度增快,表达HCV核心蛋白的Huh7-Core稳定细胞株构建成功,Core稳定表达后可促进Huh7细胞生长速度。It was to develop a hepatoma stable cell line expressing HCV core protein and to investigate the biological function of HCV core protein.HCV core genes was amplified by PCR methods,and then cloned into plasmid pSEB-3Flag by restriction enzyme digestion.Hepatoma cell line Huh7 were cotransfected with the recombinant plasmid pSEB-3Flag-Core and pAmpho.Stable cells Huh7-Core were screened with antibiotics blasticidine.HCV core gene expression was detected with RT-PCR and Western blot assay.The cell growth of stable cell line Huh7-Core was determined by MTS assay,crystal violet staining method.Results showed that Hepatoma cell lines Huh7-Core successfully express core genes,and core protein expression was verified by Western blot analysis.Cell proliferation rate of Huh7-Core was significantly accelerated,compared with Huh7 parental cells.A hepatoma stable cell line expressing HCV core protein has been successfully established.Stable overexpression of core protein sharply promoted hepatoma cells proliferation.

关 键 词：丙型肝炎病毒 核心蛋白 生物学功能 稳定细胞株 细胞增殖 

分 类 号：R735.7[医药卫生—肿瘤]