乙型肝炎病毒前-S1蛋白反式激活蛋白3基因的克隆化研究

Cloning of human gene 3 transactivated by PreS1 protein of hepatitis B virus

目的 应用抑制性消减杂交技术 (SSH)及生物信息学技术 (bioinformatics)筛选并克隆乙型肝炎病毒 (HBV)前 S1蛋白(Pre S1)反式激活新型靶基因 ,进一步阐明HBV感染相关疾病的发病机制。方法 以HBV前 S1蛋白表达质粒pcDNA3 .1(-) PreS1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HBV前 S1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 ,因其可以被前 S1蛋白反式激活 ,故命名为前 S1反式激活蛋白 3 (PS1TP3 ) ,已在GenBank中注册 ,注册号 :AY44 62 74。PS1TP3基因的编码序列全长为 1173个核苷酸 (nt) ,编码产物由 3 90个氨基酸残基 (aa)组成。结论 HBV前 S1蛋白在HBV进入宿主细胞的过程中起着重要的作用 ,最近的研究表明前 S1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 ,从而改变宿主正常的免疫应答水平 ,引起病变。HBV前 S1反式激活新靶基因的发现 ,为进一步研究HBV前

Objective To screen and clone the target genes transactivated by hepatitis B virus(HBV) PreS1 protein and to pave the way for elucidating the pathogenesis of HBV infection.Methods Suppression subtractive hybridization (SSH) technique and bioinformatics technique were used,and the mRNA from HepG2 cells transfected with pcDNA3.1(-) PreS1 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 HBV PreS1 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 PS1TP3, from the mRNA of HepG2 cells. The sequence for the PS1TP3 gene has been deposited into GenBank, the accession number is AY426672. Conclusion HBV PreS1 is a potential transactivator. These results will pave the way for the study of the molecular mechanism of the transactivating effects of HBV PreS1 protein and the development of new therapy for chronic hepatitis B.