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1、人GITRaa27-165蛋白的原核表达及其多克隆抗体制备作者:仝佳,王胜军*,陈君,毛朝明,杨云良,杨敏,胡正军,只棣媛,许化溪【摘要】 目的:表达和纯化人GITR胞外段(hGITRaa27-165)融合蛋白,制备hGITRaa27-165融合蛋白的兔源性多克隆抗体(pAb)。方法:利用PCR从pGEMThGITR获得hGITRaa27-165编码序列,将其亚克隆至原核表达载体pQE30,构建重组表达质粒pQE30hGITRaa27-165;将阳性重组质粒转化大肠杆菌,IPTG诱导表达融合蛋白,通过Profini
2、tyIMACNiChargedResin亲和层析柱进行纯化;纯化蛋白免疫新西兰大白兔,获取多克隆抗血清并利用ProteinASepharose柱进行纯化,ELISA法检测抗体效价,Westernblot法检测抗体特异性。结果:构建了pQE30hGITRaa27-165原核表达载体,原核蛋白hGITRaa27-165蛋白以包涵体形式在大肠杆菌得到高水平表达,通过复性与亲和层析获得纯度在90%以上的hGITRaa27-165蛋白,蛋白浓度为400mg/L,制备的抗hGITRaa27-165多抗效价高达1∶1.6×105,
3、Westernblot鉴定其具有良好的特异性。结论:获得高纯度hGITRaa27-165蛋白,并制备特异性pAb,将为进一步研究hGITR的生物学功能提供实验基础。15【关键词】hGITR原核表达多克隆抗体 ProkaryoticexpressionofhumanGITRaa27-165andpreparationofitspolyclonalantibody TONGJia,WANGShengjun*,CHENJun,MAOChaoming,YANGYunliang,YANGMin,HUZhengjun,Z
4、HIDiyuan,XUHuaxi DepartmentofImmunologyandLaboratoryImmunology,SchoolofMedicalTechnology;DepartmentofOphthalmology,theAffiliatedHospital;Class3,Grade2004,LaboratoryMedicineofJiangsuUniversity,Zhenjiang212013,China [Abstract]AIM:Toexpressandpurifytheextracellu
5、larregionofhumanglucocorticoidinducedtumornecrosisfactor(hGITRaa27-165),andtoprepareandidentifythepolyclonalantibody(pAb)againstthisfusionprotein.METHODS:The429bpDNAsequenceofhGITRaa27-165wasobtainedfrompGEMThGITRbyPCRandthenitwasinsertedintopQE30plasmidtoconst
6、ructtheprokaryoticexpressionplasmidpQE30hGITRaa27-165.pQE3015hGITRaa27-165wastransformedintoE.coli.ThetargetfusionproteinwasexpressedwiththeinductionofIsopropylβD1thiogalactopyranoside(IPTG)andpurifiedbyNi2+NTAaffinitychromatographycolumn.Theantiserumagain
7、sthGITRaa27-165wasobtainedfromtherabbitsimmunizedwithhGITRaa27-165.ThetiterofpAbwasdetectedbyELISAandthespecificityofpAbwasidentifiedbyWesternblot.RESULTS:TheprokaryoticexpressionplasmidpQE30GITRaa27-165wasconstructedsuccessfully.Thecultureconditioninwhichthetar
8、getfusionproteinwashighlyexpressedwasfoundout:theoptimalconcentrationofIPTGwas0.5mmol/L,theculturetemperaturewas30℃andtheculturetimewas6h.TheGITRaa27-165fusion