摘要
[目的 ]纯化抗乙酰胆碱受体单链抗体 ,为测定其特异性准备材料 .[方法 ]将大肠杆菌表达的单链抗体 (含有 6xHis及c myc标记肽 )经Ni nitrilotriaceticacid(Ni NTA)纯化 ,以十二烷基硫酸钠 聚丙烯酰胺凝胶电泳和应用鼠抗c myc单克隆抗体的免疫印迹试验鉴定纯化产物 ,并根据吸光度值测定表达产物的含量 .[结果 ]在纯化产物中发现大小约为 36KD的单链抗体 ,杂质蛋白带较少 ,表达量约为30 μg/L .[结论 ]用Ni NTA纯化带有 6xHis标记肽的单链抗体的方法简单、纯度高 ,以大肠杆菌表达抗乙酰胆碱受体单链抗体时产量低 .
OBJECTIVE?To prepare the purified single ch ain variable fragment (ScFv) of antibody directed against acetylcholine rece ptor for further determination of its specificity.?METHODS?The ScFv, bearing c-myc and 6?xHis tags and expressed in E. coli, were purifi ed by using Ni-nitrilotriacetic acid(Ni-NTA). The purified products were checked b y sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blot with using mouse anti-c-myc monoclonal antibody. The expresse d yie ld was determined based on the result of purification.?RESULTS?The purified products of expressed ScFv were found to be 36?KD in size, and the expressed yield was 30?μg/L culture medium.?CONCLUSION?T he method of using Ni-NTA for the purification of a ScFv bearing 6?xHis ta gs i s simple, and the purified products are pure. The yield of ScFv, directed agains t acetylcholine receptor, expressed by bacteria E. coli is low.
出处
《延边大学医学学报》
CAS
2004年第2期79-83,共5页
Journal of Medical Science Yanbian University
基金
国家自然科学基金 (30 36 0 10 0 )
教育部留学回国人员科研启动基金
关键词
抗体
受体
胆碱能
纯化
antibodies
receptors, cholinergic
purification
