应用抑制性消减杂交技术克隆乙型肝炎病毒前-S1蛋白反式激活的相关基因

Cloning of genes transactivated by pre-S1 protein of hepatitis B virus using suppression subtractive hybridization technique

目的 应用抑制性消减杂交 (SSH)技术构建乙型肝炎病毒 (HBV)前 S1蛋白 (pre S1)反式激活的相关基因cDNA消减文库 ,克隆前 S1反式激活相关基因 ,以期发现前 S1蛋白反式激活作用的靶位点 ,为阐明前 S1蛋白的反式激活作用的分子生物学机制 ,以及HBV相关肝细胞癌 (HCC)形成的分子生物学机制开辟新的研究方向。方法 以HBV前 S1表达质粒pcDNA3 .1(-) preS1转染HepG2细胞 ,以空载体pcDNA3 .1(-)为对照 ;制备转染后的细胞裂解液 ,提取mRNA并逆转录为cDNA ,经RsaⅠ酶切后 ,将实验组cDNA分成两组 ,分别与两种不同的接头衔接 ,再与对照组cDNA进行两次消减杂交及两次抑制性PCR ,将产物与pGEM Teasy载体连接 ,构建cDNA消减文库 ,并转染大肠杆菌DH5α进行文库扩增 ,随机挑选克隆PCR扩增后进行测序及同源性分析。结果 文库扩增后得到 67个阳性克隆 ,经菌落PCR分析 ,得到 5 1个 2 0 0～ 10 0 0bp插入片段。对所得片段测序 ,并进行同源性分析 ,获得 3 2种编码基因 ,其中 2 6个为已知功能基因 ,另外 6个为未知功能序列 ,可能是前 S1蛋白反式激活新的靶基因。结论 成功构建HBV前 S1反式激活的相关基因cDNA消减文库 ,为今后进一步分析、研究病毒蛋白的致病机制奠定基础。为进一步阐明前

Objective To construct a subtractive cDNA library of genes transactivated by pre S1 protein of hepatitis B virus (HBV) using suppression subtractive hybridization(SSH) technique and clone genes associated with transactivation,and to pave the way for elucidating the pathogenesis of hepatitis B virus(HBV) infection.Methods Suppression subtractive hybridization technique and bioinformatics technique were used,the mRNA from HepG2 cells transfected with pcDNA3.1(-) preS1 and pcDNA3 1(-) empty vector was isolated,respectively;cDNA was synthesized.After digestion with restriction enzyme RsaⅠ ,cDNA fragments were obtained.Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2,respectively.After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR,amplified cDNA fragments were subcloned into pGEM Teasy vectors to set up the subtractive library.Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.Results The subtractive library of genes transactivated by pre S1 was constructed successfully.The amplified library contains 67 positive clones.Colony PCR showed that these clones contain 200～1 000bp inserts.Sequence analysis was performed in 51 clones randomly,and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank,altogether 32 coding sequences were gotten,which consisted of 26 known and 6 unknown ones.Sequence analysis suggested that six novel cDNA sequences might be target genes transactivated by pre S1 protein.Conclusion The obtained sequences may be target genes transactivated by HBV pre S1 protein among which some genes coding proteins involved in cell cycle regulation,metabolism,immunity and cell apoptosis.This finding brought some new clues for studying the biological functions of pre S1