人F1F0ATP合成酶β亚基原核表达、纯化及抗体制备.doc

人F1F0ATP合成酶β亚基原核表达、纯化及抗体制备.doc:..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(+),(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Westernblot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(binanthATP5B,rhATP5B)%,%,%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,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,:ThecodingsequenceofhATP5BmaturepeptidewasamplifiedbyRTPCRfromhumanumbilicalveinendothelialcells(HUVEC)andthenclonedintoprokaryoticexpressionvectorpET28a(+).(DE3)+metalchelatingchromatograph,:pletelyconcordantwiththeoriginalsequence(NM_001686,hATP5B'essionnumber)・SDSPAGEshowedthattherelativemolecularmasses(Mr)oftheexpressed,purified,andrefoldedproductswereaboutMr55000,ordancewiththepredicted・binanthATP5B(rhATP5B)%ofthetotalbacteriaprotein,%,%.:640000,:hATP5Bexpressedinprokaryoticsystemhasstrongimmunogenicity,andthepolyclonalantibodywithhightiterandspecificitywasobtainedfromimmunizationofrabbits