丙型肝炎病毒非结构蛋白NS3反式激活基因1的克隆化研究

Cloning and identification of human gene 1 transactivated by nonstructural protein 3 of hepatitis C virus

目的 应用抑制性消减杂交技术 (SSH )及生物信息学技术 (bioinformatics)筛选并克隆丙型肝炎病毒非结构蛋白 3(NS3 )反式激活新型靶基因 ,进一步阐明HCV感染相关疾病的发病机制。方法 以HCVNS3蛋白表达质粒pcDNA3 1(-) NS3转染HepG2细胞 ,以空载体pcDNA3 1(-)为平行对照 ,提取mRNA并进行抑制性消减杂交分析。并应用分子生物学技术 ,结合生物信息学技术 ,克隆HCVNS3反式激活作用的新的靶基因。结果 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。从HepG2细胞提取总RNA ,以逆转录多聚酶链反应 (RT PCR)技术扩增获得该新基因的全长序列 ,并测序证实 ,因其可以被NS3蛋白反式激活 ,故命名为NS3TP1,已在GenBank中注册 ,注册号为AY116969。NS3TP1基因的编码序列全长为 193 2个核苷酸 (nt) ,编码产物由 64 2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 ,HCVNS3反式激活作用的新靶基因的发现 。

Objective To screen and clone the target genes transactivated by hepatitis C virus (HCV) nonstructural protein 3 (NS3) and to pave the way for elucidating the pathogenesis of HCV infection.Methods Suppression subtractive hybridization (SSH) technique and bioinformatics technique were used,and the mRNA from HepG2 cells transfected pcDNA3 1(-) NS3 and pcDNA3 1(-) empty vector was isolated,respectively,and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups.The coding gene transactivated by HCV NS3 was cloned by bioinformatics methods.Results The obtained sequences were searched for homologous DNA sequence from GenBank,one of which was a new gene with unknown function.The new gene with no homology with known genes in this database was confirmed and electric polymerase chain reaction (PCR) was conducted for the cloning of the full length DNA for the new gene and in conjunction with Kozak role and the exist of polyadenyl signal sequence.The reverse transcription PCR (RT PCR) was used to amplify the new gene,named as NS3TP1,from the mRNA of HepG2 cells.The sequence of the NS3TP1 gene has been deposited into GenBank the accession number is AY116969.Conclusion HCV NS3 is a potential transactivator.These results will pave the way for the study of the molecular mechanism of the transactivation effects of HCV NS3 protein and the development of new therapy for chronic hepatitis C.