摘要
目的:应用基因芯片技术.检测丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)反式激活基因NS5ATP3的表达对肝母细胞瘤细胞HepG2基因表达谱的影响.进一步阐明NS5ATP3蛋白可能的分子生物学功能.方法:没计并合成NS5ATP3基因序列特异性的引物,应用聚合酶链反应(PCR)技术扩增NS5ATP3蛋白编码基因片段,以常规的分子生物学技术将获得的NS5ATP3编码基因片段克隆到TA载体中进行核苷酸序列的测定,构建真核表达载体pcDN.A3.1(-)-NS5ATP3.以脂质体转染肝母细胞瘤细胞系HepG2,提取mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1(-)的HepG2细胞进行cDNA芯片分析.结果:构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误.提取高质量的mRNA,逆转录为cDNA,进行DNA芯片技术分析.在1152个基因表达谱的筛选中,发现有6个基因表达水平显著上调,18个基因表达水平显著下调.结论:应用基因表达谱芯片技术成功筛选了NS5ATP3转染细胞后差异表达基因,为进一步阐明NS5ATP3蛋白可能的生物学功能提供依据.
AIM: NS5ATP3 obtained from suppression subtractive hybridization screeening is a novel gene transactivated by nonstructural protein 5A (NS5A) of hepatitis C virus (HCV), which possesses unknown function. To study the difference in gene expression in human hepatoblastoma cell line HepG2 cells transfected with NS5ATP3-expressing plasmid and further elucidate its potential molecular biological function, we compared the differentially expressed genes between the HepG2 transfected by pcDNA3.1(-)-NS5ATP3 and pcDNA3.1(-), respectively by cDNA microarray technique. METHODS: Sequence specific primers were designed and synthesized and the NS5ATP3 DNA fragment was amplified with polymerase chain reaction (PCR) technique. The expressive vector of pcDNA3.1(-)-NS5ATP3 was constructed by routine molecular biological methods. cDNA microarray technology was employed to detect the mRNA from the HepG2 cells transfected with pcDNA3.1(-)-NS5ATP3 and pcDNA3.1(-), respectively using lipofectamine. RESULTS: The expressive vector has been constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis. High quality mRNA and cDNA were prepared and successful microarray screening was conducted. The scanning results indicated that among 1 152 genes which were gotten from gene expression profile analysis, there were 21 differences in which 6 genes were up-regulated and 18 genes were down-regulated in NS5ATP3-expressing HepG2 cells. These genes differentially regulated by NS5ATP3 included human genes encoding proteins involved in cell signal transduction, cell apoptosis, cell proliferation and differentiation. CONCLUSION: cDNA microarray technology is successfully used to screen the genes differentially expressed in NS5ATP3-expressing HepG2 cells, which brings some new clues for studying the potential molecular mechanism of NS5ATP3 protein.
出处
《世界华人消化杂志》
CAS
2004年第2期306-310,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金攻关项目
No C03011402
No.C30070689军队"九
五"科技攻关项目
No.98D063军队回国留学人员启动基金项目
No.98H038军队"十
五"科技攻关青年基金项目
No.01Q138军队"十
五"科技攻关面上项目
No 01MB135~~
