应用抑制性消减杂交技术克隆PS1TP1的反式激活基因

Screening of the target genes transactivated by PS1TP1 protein with 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（preS1）反式激活蛋白1（PS1TP1）的相关基因cDNA消减文库,克隆PS1TP1反式激活相关基因,以期发现PS1TP1蛋白反式激活作用的靶位点.方法 以PS1TP1表达质粒PcDNA3.1（-）-PS1TP1转染HepG2细胞,以空载体pcDNA3.1（-）为对照;提取mRNA并逆转录为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.结果 文库扩增后得到90个阳性克隆,随机挑选43个克隆测序,并进行同源性分析,获得12种编码基因,其中10个为已知功能基因,另外2个为未知功能序列.结论 成功构建PS1TP1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.为进一步研究PS1TP1蛋白的功能及其在HBV感染中的分子生物学机制提供理论依据和研究方法.

Objective To construct a subtractive cDNA library of genes transactivated by PS1TP1 protein with suppression subtractive hybridization （ SSH） technique. Methods Suppression subtractive hybridization technique and bioinformatics technique were used, the mRNA from HepG2 cells transfected with pcDNA3. 1（-）-PS1TP1 and pcDNA3. 1 （-） empty vector was isolated, respectively; cDNA underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Results The subtractive library of genes transactivated by PS1TP1 was constructed successfully. Sequence analysis was performed in 43 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 12 coding sequences were gotten, which consisted of 10 known and 2 unknown ones. Conclusion The obtained sequences may be target genes transactivated by PS1TP1 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity and cell apoptosis. This finding brought some clues for studying the biological functions of PS1TP1.