【doc】阿尔茨海默病患者β淀粉样肽40人单链抗体在大肠杆菌的表达和纯化.docVIP

阿尔茨海默病患者β淀粉样肽40人单链抗体在大肠杆菌的表达和纯化 114 中国临床康复第7D卷第2明20o6~01—15出版 ChineseJournalofClinicalRehabilitation,January152006VoL10.2 ? 基础研究? 阿尔茨海默病患者p淀粉样肽4O人单链抗体在大肠杆菌的表达和纯化 蔡炯,李方,王世真,郑连芳,蒲圻 中国协和医科大学,中国医学科学院北京协和医院核医学科,北京市 l0o730 蔡炯☆,男,1972年生,贵州省仁怀县人,汉族,20o3年中国协和医科 大学毕业,博士,助理研究员,主要从事阿尔茨海默病药物治疗的研 究.jiongcai@ 国家自然科学基金资助(3050o573) 中图分类号:R7.49.1文献标识码:A文章编号:1671—5926(2006)02～0l14_o3 收稿日期:2005～12-09修回日期:2005—12—19(05—50—9—7372/Y?L) Expressionandpurificationofhumansingle?chainantibody againstamyloidbeta40fromEscherichiacoliofpatientswitll AIzheimerdisease CaiJiongLiFang,WangShi—zhenZhengLion—fang,PuQi DepartmentofNuclearMedicin~BeijingUnionHospitalPekingUnionMedical Collegeamp;ChineseAcademyofMedicalSciences.Beijingl0o730.China CaiJiong☆.Doctor.Assisthntinvestigator,DepartmentofNuelear Medicine,BeijingUnionHospital,PekingUnionMedicalCollegeamp;Chinese AcademyofMedicalSciences,Beijing100730,Chinaiiongcai@ Supportedby:agrantfromNationalNaturalScienceFoundationof China,No Received:2005～12—09Accepted:2005—12—19 Abstract AIM-Toexpressandpurifyhumansingle—chainantibodyagainstamyloid Bpeptide40frombacteriasystemforpassiveimmunizationtoAlzheimer diseasepatients.whi晶representedapromisingtherapeuticapproach. M哐THoDS:TheexperimentwascarriedoutintheLaboratoryofNuelear MedicinebetweenAugust20o4andMarch20o5.Hunk3.nsingle—chain antibodygenefromaphagedisplaylibrarywasclonedintopET22b(+) plasmidexpressionvectorandtransformedcompetitiveBL2l—goldcells. Afterinducingthetransformedcellswith0.5mmol/Lisopropyl—B—D— thiogalactosidefIPTG).Theinsolublepelletwasdissolvedinbufferand loadedtopre—equilibratedNi一NTA—resin.Thebindingpioteinwasre— naturedandelutedfromtheresinwithimidazole.Theproteinelutedwas identifiedbyWesternblottingandenzyme—linkedimmunoadsordentassay (EL1SA1withmousemonoclonalantibodyandHRP—labeledgoatanti—mouse antibody.Concentrationofthehumansingle—chainantibodywas determinedbyBranfordmethod. RESULTS:(Anti—amyloidB40humansingle—chainantibodygene consistedofabout750bpandtherelativemolec