丙型肝炎病毒F蛋白反式激活基因2的克隆化研究

Cloning of Human Gene 2 Transactivated by F Protein of Hepatitis C Virus

目的:应用抑制性消减杂交(SSH)技术及生物信息学(bioinformatics)技术筛选并克隆丙型肝炎病毒(HCV)F蛋白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HCVF蛋白表达质粒pcDNA3.1(-)F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。应用分子生物学技术,结合生物信息学技术,克隆HCVF蛋白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(RTPCR)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F蛋白反式激活,故命名为F蛋白反式激活蛋白2(HCVFTP2),已在GenBank中注册,注册号:AY740522。HCVFTP2基因的编码序列全长为177个核苷酸(nt),编码产物由58个氨基酸残基(aa)组成。结论:HCVF蛋白在病毒的自然感染过程中有明显的表达,最近的研究表明蛋白还具有反式激活的作用,上调宿主细胞某些基因的表达,可能参与HCV致癌。HCVF反式激活新靶基因的发现,为进一步研究HCVF蛋白的分子生物学机制和探索新型治疗技术奠定了基础。

Objective： To screen and clone the target genes transactivated by hepatitis C virus （HCV） F protein and to pave the way for elucidating the pathogenesis of HCV infection. Methods： Suppression subtractive hybridization （SSH） technique and bioinformatics technique were sued, and the mRNA from HepG2 cells transfected with pcDNA3.1 （ - ） -F 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 F was cloned by bioinformatics methods.Results： The obtained sequences were searched for homologous DNA sequences 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 exist of polyadenyl signal .sequence. The reverse transcription PCR （RT-PCR） was used to amplify the new gene, named as HCV FTP2, from the mRNA of HepG2 cells. The sequence for the HCV FTP2 gene has been deposited into GenBank, the accession number is AY740522. Conclusion： HCV F may be a potential transactivator. These results will pave the way for the study of the molecular mechanism of the transactivating effects of HCV F protein and carcinogenesis of HCV infection.