丙型肝炎病毒非结构蛋白5A反式激活基因8的克隆化研究

目的 丙型肝炎病毒(HCV)的非结构蛋白5A(NS5A)是一种具有显著反式激活作用的病毒蛋白质。为了探索HCV NS5A病毒蛋白反式激活作用的新的靶基因，我们应用抑制性消减杂交(SSH)技术对于转染和未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 以HCV NSSA表达质粒peDNA3．1(-)-NSSA转染HepG2细胞，以空载体pcDNA3．1(-)为平行对照，提取mRNA并进行SSH分析。对于所获基因片段序列分析表明，其中之一为新型基因片段，与GenBank中注册的已知功能基因序列没有同源性，利用表达序列标签(EST)序列的搜索和比对，进行电子拼接。根据基因起始密码子的Kozak规则和终止密码子下游保守的多聚腺苷酸信号序列，确定新型基因序列。结果 结果从HepG2细胞提取总RNA，多聚酶链反应(RT-PCR)技术扩增获得该新基因的全长序列，测序证实，命名为NSSATP8在GenBank中注册，注册号为．AF529369.NSSATP8基因的编码序列全长为1449(nt)，编码产物由483个氨基酸残基(aa)组成。结论 HCV NS5A反式激活新型靶基因NSSATP8筛选与克隆，进一步研究HCV NS5A反式激活作用的分子生物学机制和探索新型治疗技术奠定基础。

Cloning and identification of gene 8 transactivated by hepatitis C virus non-structural protein 5A

Objective To explore the new target genes transactivatied by hepatitis C virus (HCV) non_structural protein 5A(NS5A) and pave the way for elucidating the pathogenesis of HCV infection.Methods On the base of subtractive cDNA library of genes transactivatied by NS5A protein of hepatitis C virus,the coding sequence of new gene was obtained by bioinformatics methods.Polymerase chain reaction(PCR) was conducted to amplify NS5ATA8 gene.Results The expressive vector has been constructed and approved correct.The RNA has been purified from HepG2 and HepG2 cells transfected with pcDNA3-NS5A,respectively.The cDNA derived has been subjected for microarray assay.New gene named NS5ATP8 has been cloned in combination of molecular biological and bioinformatics methods.Conclusion HCV NS5A 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 NS5A protein.These results will pave the way for study on the transactivation of HCV NS5A protein.