丙型肝炎病毒非结构蛋白NS3反式激活基因6的克隆化研究

目的 丙型肝炎病毒(HCV)非结构蛋白3(NS3)是一种具有显著反式激活作用的病毒蛋白质。为了探索HCVNS3病毒蛋白反式激活作用的新的靶基因，我们应用微矩阵(microarray)技术对于转染和末转染的肝母细胞瘤细胞系HepG2进行分析。研究结果将有助于阐明HCV感染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HCVNS3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(-)-NS3。以pcDNA3．1(-)-NS3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(bioinformatics)，克隆HCVNS3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(-)-NS3，经过限制性内切酶作图分析和核苷酸序列分析证实正确无误。以PcDNA3．1(-)-NS3转染HepG2后提取总RNA，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NS3反式激活的新型靶基因，命名为NS3TP6，新基因的编码基因序列全长为414个核苷酸(nt)，编码产物由138个氨基酸残基(aa)组成。结论 HCVNS3是一种典型的病毒基因组编码的具有反式激活作用的蛋白。微矩阵技术是分析基因表达谱变化的有效和高通量技术。发现了HCVNS3反式激活作用的新的靶基因，并成功克隆其中的NS3TP6，为阐明HcvNs3蛋白的反式激活作用及其机制，开辟了新的研究方向。

Identification and characterization of gene 6 transactivated by hepatitis C virus non-structural protein 3 with DNA microarray assay

Objective Hepatitis C virus(HCV) non structural protein 3(NS3) is a potential transactivator.In order to explore the new target genes transactivated by HCV NS3,we conducted microarray assay on the hepatoblastoma HepG2 and HepG2 transfected by NS3 expressive vector.The results will pave the way for elucidating the pathogenesis of HCV infection.Methods Sequence specific primers were designed and synthesized according to the HCV H strain of virus sequence.Polymerase chain reaction(PCR) was conducted to amplify the NS3 coding gene for the construction of expressive vector pcDNA 3.1 (-) NS3.Hepatoblastoma cell HepG2 was transfected with plasmid DNA of pcDNA3.1(-) NS3,and total RNA was purified from it.Reverse transcribed cDNA were subjected for microarray assay.The coding gene transactivated by HCV NS3 was cloned by bioinformatics methods.Results The expressive vector has been constructed and approved correct.The RNA has been purified from HepG2 and HepG2 cells transfected with pcDNA3.1(-) NS3,respectively.The cDNA derived has been subjected for microarray assay.New named NS3TP6 has been cloned in combination of molecular biological and bioinformatics methods.Conclusion HCV NS3 is a potential transactivator.Microarray is an efficient and convenient method for analysis of differentially expressed genes.A new gene has been recognized as the new target transactivated by HCV NS3 protein.These results pave the way for study on the transactivation of HCV NS3 protein.