丙型肝炎病毒非结构蛋白5A反式激活基因11的克隆化研究

目的 为了探索丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)病毒蛋白反式激活作用的新的靶基因，我们应用基因芯片技术对于转染和末转染的肝母细胞瘤细胞系H叩G2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pBRTN-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载体中进行核苷酸序列的测定，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（bioinformatics)，克隆HCVNS5A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苷酸序列分析证实正确无误。以PCDNA3．1(-)-NS5A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NS5A反式激活的新型靶基因，命名为NS5AWll，新基因的编码基因序列全长为1257个核苷酸(nt)，编码产物由418个氨基酸残基(aa)。结论 HCVNS5A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。基因芯片技术是分析基因表达谱变化的有效和高通量技术。发现了HCVNS5A反式激活作用的新的靶基因，这一发现，为阐明HCVNS5A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 11 transactivated by hepatitis C virus non-structural protein 5A with DNA microarray

Objective To explopre the new target genes transactivated by HCV NS5A,we conducted microarray assay on the hepatoblastome HepG2 and HepG2 transfected by NS5A expressive vector.Methods Sequence specific primers were designed and synthesized according to the HCV H strain of virus sequence.Polymerase chain reaction (PCR) was cinducted 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 The expressive vector has been constructed and approved correct.The RNA has been isolated from HepG2 and HepG2 cells transfected with pcDNA3 1(-) NS5A,respectively.The cDNA derived has been subjected to microarray assay.New gene named NS5ATP11 has been cloned in combination of molecular biological and bioinformatics methods.Conclusion HCV NS5A is a potential trancactivator.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 NS5A protein.These results pave the way for the study on the transactivation of HCV NS5A protein.