一种基于hiv-1 tat蛋白质转导结构域细胞内转导系统的成功改建

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1、一种基于HIV-1TAT蛋白质转导结构域细胞内转导系统的成功改建作者：郭爱华；刘志锋；孙学刚；李海玉；邓鹏；姜勇　　(南方医科大学病理生理学教研室/广东省功能蛋白质组学重点实验室，广东广州)摘要：目的  探索本室构建的pET14b-His-TAT-Flag重组载体表达的融合蛋白在细胞中转导效率低的原因，建立高效的细胞内转导系统。方法  通过PCR方法去除原有载体中的Flag序列，在改建正确的pET14b-His-TAT重组载体基础上插入EGFP编码序列，将经酶切、DNA测序鉴定正确的pET14b-His-TAT-EGFP质粒转化E.coliBL21(DE3)，经IPTG诱导表达、SDS-

2、PaGE鉴定正确，再经蛋白透析、过滤处理。将His-TAT-EGFP融合蛋白加入ECV304细胞中，用荧光显微镜观察。结果  重组质粒pET14b-His-TAT融合表达载体和pET14b-His-TAT-EGFP重组载体经酶切、DNA测序鉴定证实构建成功。表达纯化出了高纯度His-TAT-EGFP融合蛋白并具有较高细胞内转导活性。结论成功改建pET14b-His-TAT-Flag重组载体，正确构建pET14b-His-TAT-EGFP载体，His-TAT-EGFP融合蛋白高效表达，并具有明确的细胞内转导活性。关键词：HIV-1反式激活因子；蛋白质转导结构域；融合蛋白；细胞内转导中图分类

3、号：Q291  文献标识码：A  文章编号：1673-4254(2006)05-0545-04ReconstructionofanintracellulartransductionsystembasedonHIV-1TATproteintransductiondomainGUOAi-hua；LIUZhi-feng；SUNXue-gang；LIHai-yu；DENGPeng；JIANGYongDePartmentofPathophysiology/KeyLaboratoryofFunctionalProteomicsofGuangdongProvince,SouthernMedicalUn

4、iversity,Guangzhou,ChinaAbstract:ObjectiveToexplorethereasonsforthelowintracellulartransductionefficiencyofapreviouslyconstructedHis-TAT-Flagrecombinantproteinandestablishamoreefficienttransductionsystem.MethodsTheFlagtagofpET14b-His-Tat-FlagvectorwasdeletedwithPCRmutantkit,andenhancedgreenfluore

5、scentprotein(EGFP)codingsequencewasinsertedintothenewpET14b-His-TATrecombinantvector.EnzymedigestionandDNAsequencingwereperformedforidentificationofpET14b-His-TAT-EGFPvector,whichwasthentransformedintoE.coliBL21(DE3).AfterIPTGinduction,therecombinantproteinofHis-TAT-EGFPwasisolatedandanalyzedwith

6、SDS-PaGE.PurifiedHis-TAT-EGFPrecombinantproteinwasaddedtoECV304cellsandthefluorescencewasobservedtoevaluatethetransductionefficiency.ResultspET14b-His-TATvectorandpET14b-His-TAT-EGFPvectorweresuccessfullyconstructed,whichwasidentifiedwithenzymedigestionandDNAsequencing.His-TAT-EGFPfusionproteinwa

7、sexpressedandpurifiedsuccessfullyandshowedcellulartransductionactivity.ConclusionTheprokaryoticexpressionvectorhasbeensuccessfullyconstructedbymodifyingpET14b-His-TAT-Flag,andtheexpressedandpurifiedrecombinantproteinof