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1、鼠抗人cKit單克隆抗體的制備及其特性鑒定作者:劉虹辰，毛朝明，蘇曉瑜，阮錚，丁秋蘭，王學鋒，王鴻利，奚曉東【摘要】目的：鼠抗人CKit單克隆抗體（mAb）的研制及其生物學特性的鑒定。方法：利用PCR技術獲得人cKit的胞外免疫球蛋口區(qū)編碼序列，并將其亞克隆至原核表達載體PQE30,構(gòu)建出重組表達質(zhì)粒PQE30hKitD45o測序鑒定IE確后轉(zhuǎn)化M15,經(jīng)IPTG37°C誘導4h后，SDSPAGE顯示融合蛋白（表達產(chǎn)物）以包涵體形式存在。用鎳柱對融合蛋白進行純化，將純化的蛋白免疫BALB/c小鼠，采用B淋
2、巴細胞雜交瘤技術進行細胞融合，經(jīng)流式細胞術（FCM）分析和多次克隆化培養(yǎng)，篩選出特異分泌鼠抗人cKit分了mAb的朵交瘤細胞株。采用Westernblot、Ig亞型快速定性試紙法、FCM等對mAb的生物學特性進行鑒定。結(jié)杲：成功表達及純化了人cKit重組蛋白，并獲得1株持續(xù)、穩(wěn)定分泌鼠抗人cKitmAb的雜交瘤細胞株，命名為SRJloELTSA和Westernblot、FCM結(jié)果顯示了該株mAb能夠識別Kasumi口血病細胞株表面表達的天然的cKit分子，并與人正常外周血細胞無交叉反應。值得注意的是，Ka
3、sumi細胞含有一個不被SRJ1識別的亞群，雖然其表達cKit（Ab81mAb的結(jié)合正常）。結(jié)論：成功構(gòu)建了原核表達載體PQE30Kit45,并獲得高純度的人PQE30Kit45重組蛋白；成功制備了1株特異性的鼠抗人cKitmAb雜交瘤。其所分泌的抗體能特異地識別人cKit分子，并且可能成為區(qū)分細胞表血表達不同cKit分子的潛在檢測工具?！娟P鍵詞】CKit;原核重組蛋白；雜交瘤；單克隆抗體；白血病[Abstract]AIM:ToprepareanticKitmonoclonalantibodiesandc
4、haracterizetheirspecificityofepitoperecognition.METHODS:cDNAencodinghumancKitextracellulardomainwasconstructedintoaprocaryoticexpressionvectorpQE30andthecorrectnessofthereconstructedplasmidpQE30KitD45wasverifiedbysequencing?TheplasmidwastransformedintoE.c
5、oliMl5strain.Recombinant6XHispQE30KitD45wasexpressedafterinductionbyTPTGfor4h.ThenSDSPAGEresultssuggestedthattheproductsmainlyformedinelusionbodies?ThefusionproteinwasfurtherpurifiedwithNiNTAHisaffinitychromatographyandthenusedtoimmunizeBALB/cmice?Thehybr
6、idomaswereachievedbyfusingtheimmunizedspleencel1swiththeSp2/0myelomacel11ine?ThepositivecloneswerescreenedbyFCMwithCIIOhKitcells?HybidomaclonessecretinganticKitantibodieswerefurthersubclonedandinvestigatedfortheirbiologicalactivitiesbyWesternblot,rapidiso
7、typinganalysisandFCM.RESULTS:RecombinanthumancKitfusionproteinswereinvitroexpressedandpurifiedtobeusedasimmunogen.Onestablehybridomacel1line,whichcontinuouslysecretsspecificanticKitmonoclonalantibody((SRJ1))wasestablished?Thebiologicalactivitystudiesshowe
8、dthatthemonoclonalantibodyrecognizedthenaturalcKitexpressedontheKasumileukemiacellline,butfailedtobindtothenormalhumanperipheralbloodcel1s?Interestingly,thismonoclonalantibodyfailedtorecognizeasubpopulationofKasumic
