•论著*人源天然抗TNF-aScFv抗体的筛选及性质分析乔玉玲，卢卫嘉，陈继军，秦海艳，王建锋，张超，熊颖，毛晓燕兰州生物制品研究所有限责任公司第四研究室汁术省疫苗工程技术研究中心.甘南兰州730046摘要：目的以人肿瘤坏死因子(humantumournecrosisfaclor-a,humanTNF-a)^7靶抗原，从构建好的人源天然单链抗体(single-chainantibodyfragment,ScFv)噬菌体抗体库中筛选对应的ScFv抗体,验证其中和活性后测定动力学常数(kineticdissociation.KD)。方法以TNF-a梯度稀释后包被免疫管，对人源天然ScFv噬菌体抗体库进行3轮吸附-洗脱-扩增的富集筛选，制备TNF-a单克隆噬前体抗体颗粒，经E1JSA试验筛选阳性克隆后测序并分析;将正确的阳性抗体序列亚克隆到PET-26b表达裁体上,原核表达后用NiSepharose6FastFlow介质进行纯化;用MTT比色法对筛选的ScFv进行中和活性验证，并测定其中和抗体的初。结果通过3轮筛选共得到18个正确的抗体钮基酸序列，对其原核表达及纯化后得到的6株可溶性表达的抗TNF-aScFv进行中和活性验证.并对其中有中和活性的4株ScFv抗体测定KD。结论从人源天然ScFv噬菌体抗体库中成功地筛选出4株抗人TNF-a的ScFv中和抗体，其中1株ScFv抗体的3为4.80x10-8,细胞毒中和率达到48.9%,达到了药用级抗人TNF-a中和抗体的研发要求，为全分子抗体药物的构建及稳定细胞株的筛选奠定r-•定的基础。关键词：噬菌体抗体库;人肿瘤坏死因子a；筛选；中和活性;亲和力中图分类号：R392文献标志码：A文章编号：1005-5673(2018)02-0020-07DOI：10.13309/j.cnki.pmi.2018.02.005Screeningandcharacterizinganti-TNF-aantibodyfromhumannaiveScFvphagedisplaylibraryQIAOYu-ling,LUWei-jia,CHENJi-jun,QINHai-yan,WANGJian-feng,ZHANGCha。，XIONGYing,MAOXiao-YanForthDepartmentofResearch,LanzhouInstituteofBiologicalProductsCo.,Ltd.,CenterforGansuProvincialVaccineEngineeringResearch,Ixinzhou730046,GansuProvince,ChinaCorrespondingauthor：MAOXiao-yan,E-mail:maoxiaoyan2002@163.comAbstract:ObjectiveToselectanti-TNF-aScFvantibodyfromhumannaivephagedisplaylibrarybyusinghutnanTNF-aasatargetantigen,theKDofScFvantibodywasdeterminedaftertherelatedneutralizationactivityhadbeenverified.MethodsThehumannaiveScFvphagedisplaylibrarywasplannedforthreescreeningroundsofabsorbing-elution-ampli-ficationwithhumanTNF-awhichwasingradientdilutions.Theanti-TNF-amonoclonalphageantibodieswereobtainedandanalyzedbyELISA.ThecorrectsequencesofpositivecloneswereobtainedandsubclonedintopET-26b.His-TNF-afusionproteinswereexpressedinE.coliandpurifiedbyISiSepharose6FastFlowmedia.Theirsneutralizationactivitiesweretest-edbyMTTstainingandKDsweredeterminedbyBiacore.ResultsAtotalof18ScFvswereobtainedthroughthreeroundsofscreening,ofwhichaminoacidsequenceshadbeenvalidated.Thus,6ScFvswerepurifedbysolubleprokaryoticexpres-sionandtheirsneutralizationactivitieswereverified.TheKDsof4neutralizingScFvsweredetermined.ConlusionAtotalof4neutralizingScFvsagainsthumanTNF-awereobtainedfromhumannaiveScFvphagedisplaylibrary.OneoftheScFvantibodies,KDwas4.80x10*,whiletheneutralizationactivitywas48.9%.Ithasbeenachievedthedevelopmentrequirementsofthepharmaceuticalgradealx)ulantihumanTNF-aneutralizingantibody,whichlaidafoundationforthescreeningofstablecelllinesandtheconstructionofthenextfullmolecularantibodydrugs.Keywords:Phageantilxnlylibrary；Humantumournecrosisfactor-a(HumanTNF-a);Screening；Neutralizationactivity;Affinity基金项目：兰州市重大专项(2017-2-1)作者简介：乔玉玲(I982-),女,助理研究员，主耍从事基因工程抗体研究。通信作者：毛晓燕，研究员，E-mail:mHoxiaovan2002@163.com人肿瘤坏死因子a(humantumournecrosisfac-tor-a,humanTNF-a)，系由活化的巨噬细胞和单核细胞产生的细胞因子，它具有较广...