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1、細(xì)胞與分子免疫學(xué)雜志(ChinJCellMolImmuno1)2015．31(6829·論著·文章編號(hào)：1007—8738(2015)06—0829—04牛磷酸烯醇式丙酮酸羧激酶單克隆抗體的制備盛美蕾，張曉，張緯，張燕飛，王盈，戴一凡(南京醫(yī)科大學(xué)江蘇省異種移植重點(diǎn)實(shí)驗(yàn)室，南京醫(yī)科大學(xué)衛(wèi)生部抗體技術(shù)重點(diǎn)實(shí)驗(yàn)室，江蘇南京210029)[摘要]目的制備牛磷酸烯醇式丙酮酸羧激酶(PEPCK)單克隆抗體(mAb)，并對(duì)其特異性進(jìn)行初步鑒定。方法構(gòu)建含PEPCK基因的重組質(zhì)粒，將其轉(zhuǎn)化大腸桿菌，并進(jìn)行誘導(dǎo)表達(dá)，對(duì)表達(dá)產(chǎn)物PEPCK重組蛋白進(jìn)行純化，以PEPCK重組蛋白為抗原
2、，免疫BALB／c小鼠，取脾細(xì)胞與小鼠骨髓瘤細(xì)胞融合獲得雜交瘤細(xì)胞，用間接ELISA篩選陽性克隆，獲得穩(wěn)定分泌抗牛PEPCKmAb的細(xì)胞株，并用Westernblot法，免疫組織化學(xué)染色和免疫沉淀鑒定其特異性。結(jié)果成功獲得穩(wěn)定分泌抗牛PEPCKmAb的雜交瘤細(xì)胞株。Westernblot法、免疫組織化學(xué)和免疫沉淀結(jié)果表明，該抗體能與牛PEPCK蛋白特異性結(jié)合。結(jié)論成功獲得抗牛PEPCKmAb。[關(guān)鍵詞]磷酸烯醇式丙酮酸羧激酶；單克隆抗體；雜交瘤[中圖分類號(hào)]Q813．2，R392-33[文獻(xiàn)標(biāo)志碼]BSHENGMeilei，ZHANGXiao，ZHANGWei，Z
3、HANGYanfei，WANGYing，DAIYifanJiangsuProvincialKeyLaboratoryofXenotransplantation，MinistryofHealthKeyLaboratoryofAntibodyTechnique，NanjingMedicalUniversity，Nanjing210029，China【AbstractJOMectiveTopreparethemonoclonalantibodyagainstbovinephosphoenolpyruvatecarboxykinasefPEPCK)andcharacter
4、izenjsbiologicalfunctions．MetlmdsTherecombinantplasmidcontainingPEPCKgenewasconstructedandusedtotransfectEscherichiaco／i．AfterexpressioninductioninEcoli．therecombinantproteinPEPCKwaspurifiedandusedtoimmunizeBALB／cmice．AfterthespleenBcellsintheimmunizedmicewerefusedwithmurinemyelomacel
5、ls，thepositivecloneswereidentifiedandselectedbyindirectELISAfortiterdetermination．PEPCKmAbproducedbythepositivehybridomacelswasenrichedanditsbiologicalfunctionswereexaminedbyWesternblotting，immunohistochemistryandimmunoprecipitation．Results0RehybridomacelllinesteadilysecretingPEPCKmAb
6、wassuccessfullygenerated，namely3D36H．Westembloting．immunohistochemistryandimmunoprecipitationshowedthatthePEPCKmAbwasabletospecificallybindtobovinePEPCKprotein．ConclusionThebovinePEPCKmAbwaspreparedsuccessfullyandhadagoodabilityandspecificity．[Keywords]phosphoenolpyruvatecarboxykinase
7、；monoclonalantibody；hybridoma磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate正常情況下，PEPCK．C在骨骼肌中不表達(dá)或表carboxykinase，PEPCK)催化糖異生過程中的一個(gè)限達(dá)量很低。有研究表明，在骨骼肌中過量表達(dá)速反應(yīng)，即由草酰乙酸轉(zhuǎn)變?yōu)榱姿嵯┐际奖岬腜EPCK—C的PEPCK—Cmus轉(zhuǎn)基因小鼠的運(yùn)動(dòng)能力遠(yuǎn)反應(yīng)J。細(xì)胞中有2種PEPCK的同工酶，分別分遠(yuǎn)高于對(duì)照小鼠，而且PEPCK—Cmus轉(zhuǎn)基因小鼠全布在線粒體(phosphoenolpyruvatecarboxykinase-身的脂肪含量下降，不到
8、對(duì)照小鼠脂
