应用抑制性消减杂交技术克隆筛选丙型肝炎病毒F蛋白结合蛋白2反式激活基因

Cloning and Analysis of Expressed Genes Transactivated by HCVFBP2 Protein by Suppression Subtractive Hybridization

目的:应用抑制性消减杂交(SSH)技术构建新基因丙型肝炎病毒F蛋白结合蛋白2(HCVFBP2)反式激活基因差异表达的cDNA消减文库,克隆HCVFBP2反式激活相关基因。方法:以HCVFBP2表达质粒pcDNA3.1(-)FBP2转染HepG2细胞,以空载体pcDNA3.1(-)转染的HepG2细胞为对照;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多聚酶链反应(PCR),将产物与pGEMTeasy载体连接,构建cDNA消减文库,转染大肠杆菌进行文库扩增,随机挑选克隆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HCVFBP2反式激活基因差异表达的cDNA消减文库。文库扩增后选取48个克隆,进行菌落PCR分析,均得200～1000bp插入片断。挑取30个克隆进行测序,通过生物信息学分析获得28个已知功能基因序列和2个未知功能基因。结论:应用SSH技术成功构建了HCVFBP2反式激活基因差异表达的cDNA消减文库。该文库的建立为阐明HCVFBP2生物学功能提供理论依据。

Objective： To construct a subtractive cDNA library of genes transactivated by homo sapiens HCVFBP2 using suppression subtractive hybridization （SSH） technique and to clone genes associated with its transactivating function. Methods： The mRNA was isolated from HepG2 cells transfected pcDNA 3.1 （ - ） -FBP2 and pcDNA 3.1 （ - ） empty vector, respectively, to synthesize cDNA. After restriction enzyme Rsa Ⅰ digestion, small sizes cDNA were obtained. Then tester cDNA was subdivided into two portions and each ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction （PCR） twice and then was subcloned into pGEM-Teasy vectors to set up the subtractive cDNA 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 cDNA library of genes transactivated by HCVFBP2 was constructed successfully. From the resulting collection of clones, sequence information was obtained for a total of 30 distinct transcripts. Of these, 28 were found to correspong to known genes, 2 matched expressed sequence tags in public databases. Conclusion： A subtractive cDNA library of genes transactivated by HCVFBP2 using SSH technique was constructed successfully. The obtianed sequences may be target genes transactivated by HCVFBP2, which brought some new clues for studying the biological functions of HCVFBP2.