抑制性消减杂交技术筛选乙型肝炎病毒核心蛋白相互作用蛋白编码基因C1的反式调节基因_医学论文.docVIP

抑制性消减杂交技术筛选乙型肝炎病毒核心蛋白相互作用蛋白编码基因C1的反式调节基因_医学论文 抑制性消减杂交技术筛选乙型肝炎病毒核心蛋白相互作用蛋白编码基因C1的反式调节基因_医学论文 作者：蔺淑梅,张树林,成军,刘敏,郭江,张黎颖,杨媛 【关键词】 抑制性消减杂交；反式激活；克隆,乙型肝炎病毒 【Abstract】 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 subcloned into pGEMTeasy plasmid 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 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 fulllength 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. 【Keywords】 suppression subtractive