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时间:2020-05-14
《抗CD19微型双功能抗体的构建、表达及活性测定.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
1、中国癌症杂志2O1O年第2O卷第4期CHINAONCOLOGY20l0Vo1.20No.424l抗CD3/抗CD19g~型双功能抗体的构建、表达及活性测定[摘要]背景与目的:微型双功能抗体是基因工程抗体的一种形式,它具有两个抗原结合位点,可选择性募集效应细胞到靶细胞周围,介导特异性杀伤作用。本实验构建抗CD3/抗CD19微型双功能抗体(diabody)载体,表达纯化后测定其生物学活性。方法:用重叠PCR(overlapPCR)和PCR方法,构建抗CD3/抗CD19diabody载体,转化感受态大肠埃希菌进行原核表达。表达产物经抗His—tag亲和层析柱分离纯化
2、,12%SDS—PAGE电泳及Westernblot鉴定。应用间接免疫荧光和竞争性免疫荧光结合流式细胞分析技术(FACS)检测生物学活性。结果:基因重组质粒经测序证实序列正确。抗CD3/抗CD19dlabodyl~够在原核表达系统中进行可溶性表达。12%SDS—PAGE显示28×10。和26Xi0。各有一条带,Westernblot在28×10显示有条带,与预期相符。表达产物经纯化定量可达5mg/L。FACS检测抗CD3/抗CD19diabody可与CD19Raji细胞~IJCD3Jurkat细胞特异结合,并能竞争性抑制HIT3a和HIT19a与上述细胞的结合
3、活性。结论:本实验成功构建了抗CD3/抗CD19diabody,并且能够进行可溶性表达,可与CD19Raji细胞*[3CD3Jurkat细胞特异结合,为以后的抗体功能实验奠定了基础。[关键词]基因工程抗体;双功能抗体;CD3;CD19;淋巴瘤;免疫治疗中图分类号:R730.3文献标识码:A文章编号:1007—3639(2010)04-0241—06Constructionandexpressionofanti·-CD3xanti—CD19bispecificdiabodyandanalyzingitsspecificbindingactivityLIWei,F
4、ANDong-mei.CHENGXin。SHIRui—zan.LIURong,RENSi-mei.WANGMin.yANGMing(TheStateKeyLaboratoryofExperimentalHematology,InstituteofttematologyandB1oodDiseasesHospital,ChineseAcademyofMedicalSciencesandPekingionMedicalCol1eggTianjin30002~China)Correspondenceto:YANGMingE-mail:myl97o@i63.CON[A
5、bstract]Backgroundandpurpose:BispecificantibodyisaspecialpaRemofgeneticengineeringantibody.Itpossesses2antigenbindingsites,onedirectedattheefectercellsandtheotheratthetargetcellswhicharealwaysthetumorcells.Thisenablestheefectercellstorecruittothetargetseficientlyandexertcytotoxieeff
6、ect.Theexperimentwasaimedtoconstructtheexpressionvectorofanti-CD3xanti-CD19bispecificantibody,purificationandanalyzingtheactivityofthediabody.Methods:ApplyedPCRandoverlapPCRtoconstructtheexpressionvectorofanti—CD3xanti—CD19bispecificantibodies,transformeditintoE.coli16C9forexpressio
7、n.PurifiedthediabodybyHis—tagafinitychromatographyandconfirmedby12%SDS—PAGEandWesternblot.AntigenbindingactivityofthediabodywasanalyzedbyFACS.Results:TheDNAsequenceoftheexpressionvectorcontaininganti—CD3xanti—CD19fragmentwascomfirmed.Theyieldofpurifieddiabodywasupto5mg/L.Themolecula
8、rweightofdiabodywas
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