【作者】 李辉；

【导师】 孟繁平；

【作者基本信息】 延边大学 ， 病原生物学， 2005， 硕士

【摘要】 重症肌无力(myasthenia gravis,MG)是抗乙酰胆碱受体(acetylcholine receptor,AchR)抗体介导的器官特异性自身免疫病。MG患者血清中多数抗AchR抗体针对的是α亚单位上的主要免疫原区(main immunogenic region,MIR)。当致病性抗体同时与相邻两个AchR上的α亚单位相结合后,可以加速AchR的内质化作用;同时,抗体可结晶片段可以激活补体的经典途径,造成AchR的损伤。由抗AchR抗体制备的单链抗体(single chain variable fragment,ScFv)作为单价片段,不会与AchR交叉结合,又能避免激活补体;同时,ScFv与AchR α亚单位MIR结合后,可以特异性地封闭抗AchR抗体的结合位点,从而对AchR起到保护作用。 本实验将鼠源性ScFvA7基因克隆至原核表达载体pHEN2上,并将成功构建的pHEN2-ScFvA7转化入大肠杆菌E. coliHB2151中,使其在外周质中表达。之后,通过鼠抗c-myc单克隆抗体(mAb)进行斑点杂交试验,对冷硼酸钠裂解后的产物进行检测,以判定是否有目的蛋白的表达。进一步采用十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(western blotting)试验确认表达产物的分子量。经检测得知,E. coliHB2151外周质中有目的蛋白的表达,而培养液上清及对照大肠杆菌外周质和培养液上清均未发现目的蛋白的表达。在SDS-PAGE和蛋白印迹试验中发现一条分子量约为36KD的单一带,与理论估计值30.4KD相一致。ScFvA7蛋白在大肠杆菌中的成功表达,为以后的单链抗体对重症肌无力和基因治疗准备了材料。更多还原

【Abstract】 Univalent antibody fragments, single chain variable fragment(ScFv)from antibody against the main immunogenic region(MIR) of acetylcholine receptor(AchR),do not cross-link AchR molecules or bind complement; however, such fragments derived from anti-MIR monoclonal antibodies(mAbs) are able to protect the trceptor against the loss induced by intact anti-MIR mAb.After digested by endonucleases Nco I and Xho I , the ScFvA7 against MIR in mouse AchR was cloned onto vector pHEN2. Then , the recombinant plasmid pHEN2-ScFvA7 gene was transformed into E.coliHB2151 .The gene in the vector pHEN2 bearing a pelb leader and a c-myc tag were expressed of E.coliHB215.The expression was detected by dot blotting assay using a mouse anti-c-myc monoclonal antibody. Molecular weight of protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The ScFvA7 protein was found in periplasmic fraction, but no expression was discovered in culture medium ,or in periplamic fraction and culture mediun of control E.coliHB2151. Molecular weight of protein was found to be approximately 36KD by SDS-PAGE and western blotting. The ScFvA7 protein has been expressed successfully. This provides the feasibility for the further research on its application and therapy.更多还原