应用抑制性消减杂交技术筛选ns5abp37的反式调节基因

《应用抑制性消减杂交技术筛选ns5abp37的反式调节基因》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、应用抑制性消减杂交技术筛选NS5ABP37的反式调节基因作者：张雷，马清涌，孟宪魁，李康，成军【摘要】目的应用抑制性消减杂交(SSH)技术构建NS5ABP37反式激活相关基因差异表达的cDNA文库，筛选NS5ABP37蛋白反式激活相关基因。方法以表达质粒pcDNA3.1(-)NS5ABP37转染HepG2细胞，以空载体pcDNA3.1(-)为对照,从转染后的细胞裂解液中提取mRNA并合成cDNA，经RsaⅠ酶切消化后将实验组cDNA分成两组，分别衔接两种不同接头，再与对照组cDNA进行两次消减杂交及两次抑制性PCR反应;第2次PCR产物与pGEMTeasy克隆载体连接，构建c

2、DNA消减文库，并转化大肠杆菌进行文库扩增，随机挑选克隆经PCR鉴定后进行测序及同源性分析。结果成功构建了人NS5ABP37蛋白反式激活相关基因差异表达的cDNA文库。扩增后得到60个200～1000bp插入片段的克隆，随机挑选其中20个测序，并通过生物信息学分析获得其全长基因序列，结果共获得9种已知功能编码基因。结论NS5ABP37基因功能涉及细胞生长调节、信号转导和能量代谢。【关键词】抑制性消减杂交;NS5ABP37;丙型肝炎病毒;cDNA文库ABSTRACT:ObjectiveToscreenandidentifyhumangenestransactivatedbyNS5A

3、BP37byconstructingacDNAsubtractive11librarywithsuppressionsubtractivehybridization(SSH)technique.MethodsSSHandbioinformaticstechniqueswereusedforscreeningandcloningofthetargetgenestransactivatedbyNS5ABP37protein.AfterpcDNA3.1(-)NS5ABP37andpcDNA3.1(-)emptyvectortransfectedHepG2cells,themRNAwa

4、sisolatedfromthecells.SSHmethodwasemployedtoanalyzethedifferentiallyexpressedDNAsequenceinthetwogroups.AfterrestrictionenzymeRsaⅠdigestion,smallsizedcDNAswereobtained.ThentestercDNAwasdividedintotwogroupsandligatedtothespecificadaptor1andadaptor2,respectively.AftertestercDNAwashybridizedwith

5、drivercDNAtwiceandunderwenttwotimesofnestedPCR,thesecondPCRproductionwassubclonedintopGEMTeasyplasmidvectorstosetupthesubtractivelibrary.AmplificationofthelibrarywascarriedoutwithE.colistrainDH5α.ThecDNAwassequencedandanalyzedinGenBankwithBlastnsearchafterPCR.ResultsThesubtractivelibraryofge

6、nestransactivatedbyNS5ABP37wasconstructedsuccessfully.Theamplifiedlibrarycontained60positivecloneswith200-1000bpinserts.Sequenceanalysiswasperformedin20clonesrandomly,andthefulllengthsequenceswereobtainedwithbioinformaticsmethod.Altogether9codingsequenceswereobtained.ConclusionThefunctionofN

7、S5ABP3711genemaybeinvolvedincellgrowthregulation,signaltransductionandmetabolism.　　KEYWORDS:suppressionsubtractivehybridization;NS5ABP37;hepatitisCvirus;cDNAlibrary　丙型肝炎病毒(hepatitisCvirus,HCV)非结构蛋白5A(NS5A)是HCV基因组编码的一种最为重要的、具有反式激活作用等多种生物学活性的非结