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1、AFIFOATP合成酶0亚基原核表达、纯化及抗体制备作者：俞丽丽，张元军，张霞，王怡波，林建波，倪健,彭艳【摘要】目的：原核表达、纯化人F仆0ATP合成带静脉内皮细胞(HUVEC)总RNA为模板，通过RTPCR扩增hATP5B成熟肽编码序列，克隆入原核表达载体pET28a(+),转化大肠杆菌E.coliBL21(DE3)并诱导表达，表达产物用Ni2+螯合层析纯化，纯化产物用透析复性,产物用SDSPAGE和Westernblot进行鉴定。复性产物免疫家兔，制备多克隆抗体；多抗的效价用间接ELISA法检测，并用West
2、ernblot和细胞免疫荧光分析多抗的特异性和抗原结合性。结果：测序证实获得hATP5B成熟肽编码序列。SDSPAGE鉴定表明，表达、纯化、复性产物的相对分子质量(Mr)均约55000,与理论值相符。灰度扫描分析重组hATP5B(recombinanthATP5B,rhATP5B)的表达量占菌体蛋白总量的36.8%,纯化产物纯度达98.3%,复性产物纯度达99.2%oWesternblot反应阳性。多抗的平均抗体效价为1:640000,可特异识别HUVEC中的天然抗原。结论:原核表达的hATP5B具有良好的免疫原性
3、，其免疫家兔后获得的多抗具有高效价和特异性。hATP5B的原核高效表达和其抗体制备为进一步研究hATP5B的功能奠定了实验基础。【关键词】hATP5BrhATP5B原核表达多克隆抗体[Abstract]AIM:Toexpressandpurifythe0subunitofhumanFIFOATPsynthase(hATP5B)inprokaryoticsystem,andgenerateitspolyclonalantibodyinrabbits.METHODS:ThecodingsequenceofhATP5Bm
4、aturepeptidewasamplifiedbyRTPCRfromhumanumbilicalveinendothelialcells(HUVEC)andthenclonedintoprokaryoticexpressionvectorpET28a(+).TheplasmidwastransformedintoE.coliBL21(DE3)toexpresshATP5BinreponsetoIPTGinduction.ExpressedproteinwaspurifiedbyNi2+metalchelating
5、chromatograph,andrefoldedbydialysis.TheproductswereanalyzedbySDSPAGEandWesternblot.Refoldedproteinwasinjectedintorabbitstogeneratepolyclonalantibody.ThetiterofthepolyclonalantibodywasdeterminedbyindirectELISA.ThespecificityandthebindingabilityweredetectedbyWes
6、ternblotandcellimmunofluoresceneeanalysis.RESULTS:DNAsequencingconfirmedthatthecodingsequeneeofhATP5Bmaturepeptidewascompletelyconcordantwiththeoriginalsequence(NM_001686,hATP5B'sGenBankaccessionnumber)・SDSPAGEshowedthattherelativemolecularmasses(Mr)oftheexpre
7、ssed,purified,andrefoldedproductswereaboutMr55000,whichwasinaccordancewiththepredicted・GrayscaleseanningshowedthattheexpressedrecombinanthATP5B(rhATP5B)accountedfor36.8%ofthetotalbacteriaprotein,andthepurityofpurifiedproductwas98.3%,ofrefolded99.1%.Theresultof
8、Westernblotispositive.Thetiterofthepolyclonalantibodywas1:640000,anditspecificallyrecognizedthenativeantigeninHUVEC.CONCLUSION:hATP5Bexpressedinprokaryoticsystemhasstrongimmunogeni
