乙型肝炎病毒前-S2蛋白反式激活蛋白1基因的克隆化研究
Cloning of human gene 1 transactivated by pre-S2 protein of hepatitis B virus
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摘要: 目的 应用基因表达谱芯片技术及生物信息学技术筛选并克隆乙型肝炎病毒(HBV)前-S2蛋白反式激活新型靶基因,进一步阐明HBV感染相关性疾病的发病机制。方法 以HBV前-S2蛋白表达质粒pcDNA3.1(-)-PreS2转染HepG2细胞,以空载体pcDNA3.1(-)为平行对照,提取总RNA进行基因表达谱芯片分析。并克隆HBV前-S2反式激活作用的新的靶基因。结果 获得该新基因的全长序列,并测序证实,因其可以被前-S2蛋白反式激活,故命名为前S2反式激活蛋白1(PS2TP1),已在GenBank中注册,注册号:AY561706。PS2TP1基因的编码序列全长为1113个核苷酸(nt),编码产物由370个氨基酸残基(aa)组成。结论 HBV前-S2蛋白具有反式激活功能,调节宿主细胞某些基因的表达,从而改变宿主正常的应答水平,引起病变。Abstract: ObjectiveTo screen and clone the target genes transactivated by hepatitis B virus (HBV) Pre-S2 protein in order to pave away for elucidating the pathogenesis of HBVinfection.MethodsHBVPre-S2 coding DNAfragment was amplified by polymerase chainreaction (PCR) using G3767 plasmid containing the full length of HBVgenome as a template. An expressive vector of pcDNA3.1 Pre-S2 was constructed with routine molecular biological methods. HepG2 cells were transfected with pcDNA3.1 (-) and pcDNA3.1 Pre-S2, respectively, using the FuGENE6 transfection reagent. Total RNA was isolated and reverse transcribed. cDNAs were subjected tomicroarray screening with 1 152 cDNA probes.ResultsFull length sequences of the obtained genes were searched from GeneBank.One of the obtained genes was a newgene with unknown functions. The newgene that has no homology with known genes in GeneBankwas confirmed with its full length DNA cloned by polymerase chain reaction (PCR). Reverse transcription PCR (RT-PCR) was usedto amplify the new gene, PS2TP1, from mRNA of HepG2 cells (The access number of PS2TP1 gene Gen-Bank is AY561706).ConclusionHBV Pre-S2 protein is a transcriptional transactivator. The results of our study may help to find the molecular mechanismunderlying the transactivating effects of HBV Pre-S2 protein and to develop new therapies for chronic hepatitis B.
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