资源描述：
《GABARAP克隆表达及其抗体的制备与应用》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。
1、生物工程学报ChinJBiotech2008,February25;24(2):245-249journals.im.ac.cnChineseJournalofBiotechnologyISSN1000-3061cjb@im.ac.cn©2008InstituteofMicrobiology,CAS&CSM,Allrightsreserved研究报告GABARAP克隆表达及其抗体的制备与应用112121李雪旎,韩亮,王芳,黄世思,张慧,常智杰1清华大学医学院清华大学生物科学与技术系,北京1000842北京师范大学生命科学院生物医学研究所,北京100875摘要:GABARAP(G
2、ABAA-receptor-associatedprotein)是最新发现的与GABAA受体γ2亚基胞内区有相互作用的蛋白,目前的研究结果表明它可能是通过与微管相互作用辅助GABAA受体向细胞膜上运输并使受体在膜上聚集。本文中用PCR方法扩增出编码人GABARAP(hGABARAP)的cDNA片段,然后分别克隆到真核表达载体pcDNA6/HA和谷胱甘肽转移酶(GST)融合蛋白表达载体pGEX4T2中。将后者导入大肠杆菌BL21中,经过IPTG诱导后用谷胱甘肽偶联的Sep-harose-4B柱子纯化出融合蛋白GST-hGABARAP。以此蛋白作为抗原免疫家兔制备抗GABARAP的
3、抗血清,并用GST-hGABARAP耦联的NHS-activatedSepharose4柱子纯化抗体。纯化的抗体可以用于真核细胞中过量表达hGABARAP的Western和免疫染色检测。结果表明,过量表达的hGABARAP在真核细胞核和细胞质中都有表达,部分集中于核周区域。关键词:GABARAP,GST融合蛋白,抗体CloningandExpressionofHumanGABARAPandPrepara-tionofAnti-GABARAPAntibodies112121XueniLi,LiangHan,FangWang,SersurNg,HuiZhang,andZhijieC
4、hang1SchoolofMedicine,DepartmentofBiologicalSciencesandBiotechnology,TsinghuaUniversity,Beijing100084,China2BiomedicalResearchInstitute,LifeScienceCollege,BeijingNormalUniversity,Beijing100875,ChinaAbstract:GABARAP,amicrotuble-associatedprotein,isidentifiedtointeractwithGABAAreceptor.Anchori
5、ngoftheGABAAreceptortoGABARAPhelpstoclusterthereceptoratthesynapticterminiandtomediatefastsynaptictransmission.GABARAPmaymediateinteractionofgephyrinwiththeGABAAreceptortostabilizeclustersbyformingmultimericstructures.Furthermore,GABARAPandgephyrinmayplaymoreofaroleinreceptorsortingandtransp
6、orttothecellsurfacethaninanchoringtothecyto-plasm,becauseatinhibitorysynapsesGABARAPappearstoassociatewithtransportvesiclesratherthanthecellsurface.Theasso-ciationofGABARAPwithNSF(N-ethylmaleimidesensitivefactor),aproteininvolvedinintracellularvesicletransport,supportsthishypothesis.Wecloned
7、cDNAencodingfull-lengthhumanGABARAPbynestedPCRandinserteditintoeukaryonexpressionvectorpcDNA6HAandGSTfusionproteinexpressionvectorpGEX4T2.TherecombinantplasmidpGEX4T2-hGABARAPwastransformedintoE.coliBL21,fromwhichGST-hGABARAPfusionproteinwaspurifie
