丙型肝炎病毒非结构蛋白5A反式激活基因NS5ATP13的克隆化研究

目的 应用微矩阵(microauray)技术，结合生物信息学(bioinformatics)技术筛选并克隆丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)的反式激活基因，阐明授性HCV感染、肝纤维化及肝细胞癌(HCC)的等相关疾病的发病机制。方法 根据HCV-H病毒株序列设计、合成序列特异性的引物。以含有全长HCV—H株cDNA的pBRTM-3011质粒DNA作为模板，进行多聚酶链反应(PCR)扩增，获得的HCVNS5A编码基因片段克隆到TA载体中进行核昔酸序列的测定，构建真核表达载体PcDNA3．1(-)-NS5A。以pcDNA3．1(-)-NS5A转染肝母细胞瘤细胞系HepG2，提取总RNA，逆转录为cDNA后进行表达谱基因芯片分析。应用分子生物学技术，结合生物信息学技术，克隆HCVKDA反式激活作用的新的靶基因。结果 构建了真核表达载体pcDNA3．1(-)-NS5A，经过限制性内切酶作图分析和核苔酸序列分析证实正确无误。以PcDMA3．1(-)-NS5A转染HepG2后提取总MA，逆转录后进行表达谱基因芯片技术分析。应用分子克隆技术结合生物信息学技术克隆NS5A反式激活的新型靶基因，命名为NS5ATP13，在GenBank中登录，登录号为D21262。Ns5ATP13基因的编码序列全长为2103个核苷酸(nt)，编码产物由700个氨基酸残基(aa)组成。结论 丙型肝炎病毒NS5A基因产物具有显著的反式激活作用。微短阵技术是分析基因表达谱变化的自动化和高通量技术。应用这些技术，发现了HCVKDA反式激活作用的新的靶基因，将为进一步研究HCVNS5A反式激活作用的分子生物学机制及HCV相关疾病发病机制奠定基础。

Cloning and identification of gene 13 transactivated by hepatitis C virus non-structural protein 5A

Objective screen and clone the target genes transactivated by nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) using microarray and bioninformatics techniques.The results will pave the way for elucidating the pathogenesis of HCV infection,hepatic fibrosis and hepatocellular carcinoma (HCC).Methods Sequence specific primers were designed and synthesized according to the HCV H strain of virus sequenec.Polymerase chain reaction (PCR) was conducted to amplify the NS5A coding gene for the construction of expressive vector pcDNA3 1(-) NS5A.Hepatoblastoma cell line HepG2 was transfected with plasmid DNA of pcDNA3 1(-) NS5A,and total RNA was purified from it.Reverse transcribed cDNA were subjected for microarray assay.The coding gene transactivated by HCV NS5A was cloned by bioinformatics methods.Results New gene discovered has been named NS5ATP13.The sequence for the NS5ATP13 gene was deposited into GenBank,and the accession number is D21262 HCV NS5A is a potential transactivator.Microarray is an effecient and convenient method for analysis of differentially expressed genes.Conclusion These results will pave the way for the study of the molecular mechanism of the transactivation effects of NS5A protein of HCV and the pathogenesis of some diseases related to HCV by microarray.