丙型肝炎病毒非结构蛋白4A反式激活基因NS4ATP1的克隆化研究

Cloning and identification of human gene 1 transactivated by nonstructural protein 4A of hepatitis C virus

目的:筛选并克隆丙型肝炎病毒(HCV)非结构蛋白4A(NS4A)反式激活新型靶基因。方法:以HCVNS4A蛋白表达质粒pcDNA3.1(-)-NS4A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。分析筛选得到的克隆,其中之一与GenBank中注册的已知功能基因序列没有同源性,通过序列同源性比对和电子拼接,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。从转染pcDNA3.1(-)-NS4A的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,并对克隆的基因及其编码产物的序列进行分析。结果:该新基因的编码序列全长为963个核苷酸(nt),编码产物由321个氨基酸残基(aa)组成,并测序证实,命名为NS4ATP1,在GenBank中注册,注册号为AY 740521。结论:分子生物学技术与生物信息学技术相结合,发现并鉴定、克隆了HCV非结构蛋白反式激活作用的新型靶基因NS4ATP1,为进一步研究HCV非结构蛋白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。

Objective ：To screen and clone the target genes transactivated by nonstructural protein 4A （NS4A） of hepatitis C virus （HCV）. Methods：The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 （-）-NS4A and pcDNA3.1 （-） empty vector, respectively, and suppression subtractive hybridization （SSH） method was employed to analyze the differentially expressed DNA sequence between the two groups. On the base of subtractive cDNA library of genes transactivated by HCV NS4A, the coding sequence of a new gene was obtained by bioinformatics methods. The new gene with no homology with known genes in GenBank database was confirmed. The reverse transcription PCR （RT-PCR） was used to amplify the new gene using mRNA from HepG2 cell as the template. The coding sequence for the new gene was deduced according to the nucleotide sequence. Results：A new gene with unknown function is named as NS4ATP1. The nucleotide sequence of the NS4ATP1 gene and the corresponding protein-encoding amino acid have been determined, which contain 963 nt and 321 aa. The sequence of the NS4ATP1 gene was deposited into GenBank, and the accession number is AY 740521. Conclusions：NS4ATP1 gene transactivated by HCV NS4A protein was cloned and identified successfully by combining molecular biological technology and bioinformatics technique. These results will pave the way for the study of the molecular mechanism of the transactivating effects of HCV NS4A protein and the development of new therapy for chronic hepatitis C.