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Screening and cloning of target genes transactivated by human gene C1 encoding protein 1 interacting with HBV core antigen using suppression subtractive hybridization technique
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 19
- DOI:
- 10.3321/j.issn:1000-2790.2006.19.004
- Key Word:
- 抑制性消减杂交;反式激活;克隆;乙型肝炎病毒
Abstract： 目的:筛选、克隆与乙型肝炎病毒(HBV)核心抗原(HBcAg)相互作用的蛋白基因C1的反式激活基因,探索该基因可能的生物学功能. 方法:以分子生物学技术构建C1真核表达载体pcDNA3.1(-)-C1, 以表达质粒pcDNA3.1(-)-C1转染HepG2细胞,以空载体pcDNA3.1(-)转染的HepG2细胞为平行对照,制备转染后的细胞裂解液,提取mRNA并逆转录为cDNA,经Rsa I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聚合酶链反应(PCR),将产物与pGEM-Teasy载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增,随机挑选阳性克隆PCR扩增后进行测序及同源性分析. 结果:成功构建人类新基因C1反式激活基因差异表达的cDNA消减文库. 文库扩增后挑选40个阳性克隆,进行菌落PCR分析,均得到200～1000 bp插入片段,随机挑选含有插入片段的30个克隆进行测序,并通过生物信息学分析获得其全长基因序列,结果共获得18种编码基因. 结论:筛选到的cDNA全长序列,包括一些与细胞周期及代谢、肿瘤发生发展及肝脂肪变及肝纤维化发生发展密切相关的蛋白编码基因,推测了C1在体内可能存在的调控机制的线索.
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