摘要
目的应用抑制性消减杂交（SSH）技术构建丙型肝炎病毒非结构蛋白4A（HCV NS4A）转染细胞差异表达cDNA消减文库，克隆HCV NS4A蛋白反式激活相关基因。方法以HCV NS4A表达质粒pcDNA3．1（-）-NS4A转染Hep G2细胞，以空载体pcDNA3．1（-）为对照，制备转染后的细胞裂解液，提取mRNA并逆转录为cDNA，进行SSH分析。将富集的二次PCR产物与TIA载体连接，并转染大肠埃希菌进行文库扩增，随机挑取克隆，聚合酶链反应（PCR）扩增后进行测序及同源性分析。结果文库扩增后得到36个阳性克隆，经菌落PCR分析显示200～1000bp插入片段。对其中的25个片段测序，并进行同源性分析，显示18种已知基因编码蛋白和2种未知功能基因序列，包括一些与细胞周期、细胞凋亡、信号传导及肿瘤发生等细胞生长调节密切相关的蛋白编码基因，可能是NS4A反式激活靶基因。结论成功构建了HCV NS4A反式激活基因差异表达的cDNA消减文库，为进一步阐明HCV NS4A反式调节的靶基因等提供了相关的平台。
Objective To construct a subtractive cDNA library of genes transactivated by NS4A protein of hepatitis C virus with suppression subtractive hybridization technique （ SSH）. Methods The mRNA was isolated from Hep G2 cells transfectod pcDNA3.1 （-）-NS4A and pcDNA3.1 （-） empty vector, respectively, then the cDNA was synthesized. SSH method was employed to analyze the differentially expressed RNA sequence between the two groups. The twice enriched PCR products were subclonod into T/A vectors to set up the subtractive library. Amplification of the library was carried out with E. coil strain JM109. The cDNA was sequenced and analyzed in comparison with those in GenBank with Blast search after PCR. Results The amplified library contained 36 positive clones. Colony PCR showed that 36 clones contained 200-1000 bp inserts. Sequence analysis was performed in 25 clones, and the full length sequences were obtained with bioinformatics method. Altogether 20 kinds of coding sequences were achieved, which consisted of 18 kinds of known and 2 kinds of unknown ones. The obtained sequences may be target genes transactivatod by NS4A protein of HCV, among which some genes coding for proteins involved in cell cycle regulation, cell apoptosis, signal transduction pathway and tumour development. Conclusion A subtractive library of genes transactivatod by NS4A protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2006年第3期270-272,共3页
Chinese Journal of Experimental and Clinical Virology
关键词
肝炎病毒
病毒非结构蛋白质类
反式激活(遗传学)
抑制
遗传
杂交
Hepacivirus
Viral nonstructural proteins
Trans-activafion （Genetics）
Suppression, genetic
Hybridization
