卵巢癌抗独特型单链抗体6B11ScFv/mGM-CSF融合蛋白的构建、表达及活性测定【摘要】目的构建卵巢癌抗独特型单链抗体6B11ScFv和鼠GM-CSF融合蛋白(6B11mGM)，以观察其在动物体内诱导的特异性免疫反应，为卵巢癌抗独特型疫苗应用于临床提供依据。方法用DNA重组技术，将mGM-CSF连于单链抗体6B11ScFv羧基末端，构建重组质粒pET30-6B11mGM，转化大肠杆菌BL21(DE3)，IPTG诱导，以包涵体形式获高效表达，超声破碎细菌细胞获得包涵体，用8mol.L-1尿素溶解包涵体后直接稀释复性，SDS-PAGE分析蛋白纯度，ELISA分析技术和细胞增殖实验测定融合蛋白的抗体和细胞因子活性。结果复性蛋白纯度达90%以上。采用氧化型谷胱甘肽(GSSG)浓度为1mmol.L-1，还原型谷胱甘肽(GSH)浓度为5mmol.L-1，10℃复性48h，复性率达36%。表达的融合蛋白分别能与COC166-9单抗和大鼠抗小鼠GM-CSF单抗特异结合，并能刺激mGM-CSF依赖株NFS-60细胞增殖。结论以包涵体表达的融合蛋白6B11mGM保留了两种蛋白的活性，为研究融合蛋白在体内的免疫功能提供了基础。【关键词】卵巢癌；重组融合蛋白；抗体；抗独特型；粒细胞-巨噬细胞集落刺激因子；包涵体【中分类号】R392.11【文献标识码】A【文章编号】0529-1356(2000)03-226THECONSTRUCTION，EXPRESSIONANDACTIVITYEXAMINATIONSOF6B11ScFv／MURINEGM-CSFFUSIONPROTEINLIUBei,CUIHeng?,FENGJie,YEXue,LIYI,CAOShan-jin,GEHua,FUTian-yun,YAOYu,QIANHe-nian(GynecologicOncologyCenter,People'sHospital,BeijingMedicalUniversity,Beijing100044,China)【Abstract】ObjectiveToconstructexpressionvectorwhichexpressesfusionproteinofantiidiotypicsinglechainantibody6B11ScFvandmurineGM-CSF(6B11mGM)forobservingitspossibleimmunereactionsinvivo.MethodsUsingDNArecombinanttechniques,themurineGM-CSFcDNAgenewasrecombinedto6B11ScFvandtheywereclonedintoexpressionvectorpET-30a(+)toproduceinsolubleprotein.Inclusionbodiescollectedafterthebreakageofbacteriathroughsonicationweresubjectedtorepeatedlywashing.Inclusionbodiessolubilizedintheresenceof8mol.L-1ureaweredilutedwithrenaturationsolutionssothatfoldingprocesscouldbeinitiated.ThepuritywasexaminedbySDS-PAGE,ELISAandcellproliferationassaywereusedtodeterminetheactivitiesoffusionprotein.Results6B11mGMfusionproteinswereobtainedwithapurityofover90%.Theoptimumconditionswere1mmol.L-1and5mmol.L-1fortheconcentrationsofGSSGandGSH,respectively,with48hoursat10℃asrenaturedtime.Fusionproteincouldspecificallyreactwiththeprimaryanti-ovariancarcinomamonoclonalantibody(COC166-9)andratanti-mouseGM-CSFmonoclonalantibody,respectively,andfusionproteinscouldalsostimulatemurineGM-CSFdependentcelllineNFS-60cellstoproliferate.ConclusionFusionproteins6B11mGMexpressedasinclusionbodiescankeeptheactivityofboththeproteinsandprovidfoundationtoresearchtheimmunoreactionsinvivo.【Keywords】Ovariancarcinoma;Recombinantfusionproteins;Antibodies,Anti-idiotypic;Granulocyte-macrophagecolony-stimulatingfactor;Inclusionbodies本中心曾用卵巢上皮癌可溶性抗原OC166-9免疫BALB／c小鼠，制备了单克隆抗体COC166-9。经免疫组织化学染色证实该抗体对卵巢上皮癌具有较高的特异性［1］。继而用COC166-9单抗免疫小鼠制备了内影像型抗独特型抗体6B11。经免疫竞争抑制试验证实，该抗体具有模拟卵巢上皮癌初始抗原OC166-9的作用［2］，可在体内外诱导产生特异的抗卵巢癌免疫反应［3，4］。为了降低鼠源性抗体反复在体内应用时可能引起的副作用，本中心对卵巢癌抗独特型抗体6B11进行改造，构建了单链抗体6B11ScFv［5］。改型后的抗体，虽降低了鼠源性，但同时因分子量减少，免疫原性也随之下降，应用时必须与匙孔血蓝蛋白(keyholelimpethemocyanin,KLH)等偶联并加用佐剂才能引起有效的免疫反应［6］，而现在常用佐剂往往对人体有害。因此，有必要寻求一种新的佐剂。最近研究表...