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目的:应用抑制性消减杂交(SSH)技术及生物信息学(bioin formatics)技术筛选并克隆乙型肝炎病毒(HBV)全S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全S蛋白表达质粒pcDNA3.1(-)-全S转染HepG2细胞,以空载体pcDNA3.1(-)为平行对照,提取mRNA逆转录为cDNA,经RsaⅠ酶切后将实验组cDNA分成2组,分别与2种不同的接头衔接,与对照组cDNA进行二次杂交和二次PCR.并结合生物信息学技术,克隆HBV 全S反式激活作用的新型靶基因. 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.成功克隆出他的全长序列并测序证实,其可以被全S蛋白反式激活,故命名为全S反式激活蛋白1 (CSTP1),已在GenBank中注册,注册号:AY553877.CSTP1 基因的编码序列全长为945个核苷酸(nt),编码产物由315 个氨基酸残基(aa)组成. 结论:HBV全S蛋白具有反式激活蛋白1基因克隆成功. HBV全S反式激活新靶基因的发现,为进一步研究HBV全S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 new target genes of hepatitis B virus (HBV) full S transactivation by SSH and bioinformatics to further elucidate the pathogenesis of HBV infection-related diseases.Methods : HepG2 cells were transfected with the pcDNA3.1 (-) - full-length HBV full S protein and the empty vector pcDNA3.1 (-) was used as a parallel control. The mRNA was reverse transcribed into cDNA. After digestion with RsaI, the experimental group The cDNAs were divided into two groups and ligated with two kinds of different linkers, respectively, and the control group cDNAs were subjected to the second hybridization and the second PCR, and combined with bioinformatics technology to clone a novel target gene of HBV full S transactivation.Results: Sequence analysis of the obtained gene fragment showed that one of them was a novel gene fragment, and the full-length sequence was successfully cloned and sequenced to confirm that it can be transactivated by the whole S protein so named as full S transactivator 1 (CSTP1 ) Has been registered in GenBank with the accession number AY553877. The full-length coding sequence of the CSTP1 gene is 945 nucleotides (nt), and the encoded product consists of 315 amino acid residues (aa) .Conclusion: Transactivator 1 gene was cloned successfully The discovery of target genes lay the foundation for further study on the molecular biological mechanism of HBV whole S and explore new therapeutic techniques.