摘要
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200～1000bp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.
AIM： To clone and identify human genes transactivated by human gene C1 encoding protein C1 interacting with hepatitis B virus （HBV） core antigen by constructing a cDNA subtractive library with suppression subtractive hybridization （ SSH ） technique. METHODS ： SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by C1 protein, The mRNA was isolated from HepG2 cells transfected with pcDNA3. 1 （ - ）-C1 and pcDNA3, 1 （ - ） empty vector, respectively, SSH method was employed to analyze the differentially expressed cDNA sequence between the 2 groups. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA was divided into 2 groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent polymerase chain reaction （PCR） twice and then was subeloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DHSα. The cDNA was sequenced and analyzed in GenBank with BLAST search after PCR. RESULTS： The subtractive library of genes transactivated by C1 was constructed successfully. Colony PCR of 40 positive clones showed that these clones contained 200- 1000 bp inserts. Sequence analysis was performed in 30 clones, at random, and the full-length sequences were obtained with bioinformatics method. Altogether 18 coding sequences were gotten. CONCLUSION： The obtained sequences may be target genes transactivated by C1 among which some genes coding proteins were involved in cell cycle regulation, metabolism, tumor immunity and development, and initiation and development of liver fibrosis. This finding brought some new clues for studying the biological functions of C1.
出处
《第四军医大学学报》
CAS
北大核心
2006年第19期1741-1744,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30571649)
关键词
抑制性消减杂交
反式激活
克隆
乙型肝炎病毒
suppression subtractive hybridization
transactivation
clone
hepatitis B virus
