应用抑制性消减杂交技术克隆NS5A-TP4反式激活基因

Cloning and Analysis of Expressed Genes Transactivated by NS5A-TP4 Protein by Suppression Subtractive Hybridization

目的:应用抑制性消减杂交(SSH)技术构建人类新基因NS5A-TP4反式激活基因差异表达的cDNA消减文库,克隆NS5A-TP4反式激活相关基因,了解该基因的可能生物学功能。方法:以NS5A-TP4表达质粒pcDNA3.1(-)-TP4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,从总RNA中提取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NS5A-TP4反式激活基因差异表达的cDNA消减文库。文库扩增后得到63个白色克隆,进行菌落PCR分析,均得到200～1000 bp插入片段。挑取含有插入片段的36个克隆进行测序,并通过生物信息学分析获得20种已知功能基因序列和5个未知功能基因。结论:应用SSH技术成功构建了NS5A-TP4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NS5A-TP4生物学功能提供理论依据。

Objective: To construct a subtractive cDNA library of genes transactivated by homo sapiens NS5A-TP4 using suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivating function.Methods: The mRNA was isolated from HepG2 cells transfected pcDNA3. 1 ( - ) -NS5A-TP4 and pcDNA3. 1 ( - ) empty vector, respectively, to synthesize cDNA. After restriction enzyme Rsal 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 se-quenced and analyzed in GenBank with Blast search after PCR.Results: The subtractive cDNA library of genes transactivated by NS5A-TP4 was constructed successfully. From the resulting collection of clones, sequence information was obtained for a total of 36 distinct transcripts.Of these, 30 were found to correspond to known genes, 6 matched expressed sequence tags in public databases.Conclusion: A subtractive cDNA library of genes transactivated by NS5A-TP4 using SSH technique was constructed successfully.The obtained sequences may be target genes transactivated by NS5A-TP4, which brought some new clues for studying the biological functions of NS5A-TP4.