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

1、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         07-08-23 14:15:00     编辑：studa20            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】  A

2、IM： 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

3、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 rest

4、riction 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 subc

5、loned into pGEMTeasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5. The cDNA was sequenced and analyzed in GenBank with BLAST search after PCR. RESULTS： The subtractive library of genes transactivated by C1 was constructed suc

6、cessfully. 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 seq

7、uences 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

8、.   【Keywords】 suppression subtractive hybridization; transactivation; clone; hepatitis B virus   【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

9、，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pGEMTeasy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2001000 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cDN

10、A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.   【关键词】 抑制性消减杂交；反式激活；克隆; 乙型肝炎病毒    0引言   乙型肝炎病毒（HBV）核心抗原（HBcAg）由乙型肝炎病毒C基因的C区编码的一种结构性蛋白，在HBV的生活周期中，HBcAg,病毒mRNA和DNA聚合酶共同构成核心颗粒，在核心颗粒中完成病毒DNA的合成. HBcAg对于乙肝病毒前基因组RNA的装配、基因组DNA的合成具有重要作用，还与病毒成熟、识别包

11、膜蛋白以及病毒向细胞外释放等过程密切相关1. HBcAg特异性CD4+ T细胞免疫应答是清除HBV的重要反应2. 我们利用酵母双杂交技术对白细胞cDNA文库中与HBcAg相互作用的蛋白进行研究，发现了一种能与HBcAg结合的未知功能蛋白，将其编码该蛋白的基因命名为C1，GenBank收录AY555145,利用生物信息学技术确定其开放读码框架（ORF），并对其进行了克隆化研究，顺利得到了C1基因编码序列. 其编码区为366个核苷酸(nt),编码121个氨基酸残基，我们利用抑制性消减杂交技术（SSH）构建C1作用于肝母细胞瘤细胞系HepG2细胞的反式调节cDNA消减文库，筛选差异表达的基因片段，并

12、应用生物信息学进行分析，为进一步了解C1的功能及其探讨HBV感染的发病机制提供理论依据.   1材料和方法   1.1材料HepG2细胞及感受态大肠杆菌DH5为本室保存，pcDNA3.1（-）真核表达载体购自Invitrogen公司；FuGENE6转染试剂购自Roche公司，mRNA Purification试剂盒购自Amersham Pharmacia Biotech公司，PCRSelect cDNA Subtraction试剂盒，50×PCR Enzyme Mix， Advantage PCR Cloning试剂盒购自Clontech公司，

13、High Pure PCR Product Purification试剂盒购自Boehringer Mannheim公司，T7，SP6通用引物及pGEMTeasy载体购自Promega公司. 真核表达质粒pcDNA3.1(-)c1由本室构建. DNA序列测定由上海联合基因公司完成.   1.2方法   1.2.1真核表达载体的细胞转染及mRNA提取用FuGENE6脂质体转染试剂将2 g的 pcDNA3.1(-)c1及pcDNA3.1(-)空载体分别转染35 mm平皿HepG2细胞，48 h后收获细胞. 使用QuikPreP mico mRNA Purification试剂盒从HepG2细胞中直接提取重组表达质粒及空载体的HepG2细胞的mRNA，经琼脂糖凝胶电泳及分光光度计分别进行定量分析.   1.2.2消减杂交文库的建立采用Clontech公司的PCRSelect cDNA Subtraction Kit，常规SSH方法按说明书进行：以转染了重组表达质粒及空载体的HepG2细胞mRNA为模板逆转录合成双链cDNA（dscDNA），并分别标记为Tester和Driver