抗NMDAR1表位多肽的单克隆抗体的制备及鉴定
周游1，相琳1，张冬梅2
基金项目：辽宁省博士科研启动基金（20101027）；高等学校博士学科点专项科研基金（2010210512003） 作者简介：周游 通信联系人：张冬梅（1972年-），女，副教授，生理学.

（1. 大连大学职业技术学院； 5 2. 大连医科大学生理学教研室， 大连 116044）
摘要：目的 制备针对NMDAR1特异性B细胞表位的单克隆抗体，并对其特性进行鉴定。方法 分别采用匙孔血蓝蛋白(KLH)偶联多肽和多抗原肽(MAP)免疫Balb/c鼠，取血清效价高者脾细胞与SP2/0骨髓瘤细胞进行融合，采用间接ELISA筛选阳性杂交瘤细胞，并经有限稀释法克隆化培养获得杂交瘤细胞株，鉴定抗体类型，以Western blot鉴定单抗的特异性，用免疫荧光对单抗进行初步应用研究。结果 用KLH偶联多肽免疫小鼠的方法获得一株能稳定分泌抗NMDAR1特异B细胞表位的单克隆抗体的杂交瘤细胞，抗体类型为IgM。蛋白印迹和免疫荧光检测显示，该单抗能特异性的识别SD鼠脑组织中的相应抗原。而用MAP免疫小鼠的方法未能筛选出阳性单克隆抗体。 结论 KLH偶联多肽的免疫原性要强于MAP，用KLH偶联多肽免疫动物更容易获得针对表位多肽的抗体。针对NMDAR1特异性B细胞表位的单克隆抗体的成功制备，为进一步研究NMDAR1的功能和免疫治疗癫痫、脑缺血等神经系统疾病提供了研究工具。
关键词：NMDAR1；B细胞表位；单克隆抗体；治疗性抗体
中图分类号：R338
Preparation and characterization of monoclonal antibody against NMDAR1 epitope peptide
ZHOU You1, XIANG Lin1, ZHANG Dongmei2
(1. Vocational and Technical College of Dalian University;2. Physiological Department of Dalian uniersity, Dalian 116044)
Abstract: To prepare monoclonal antibody (mAb) against NMDAR1 specific B cell epitope and characterize its properties, Balb/c mice were immunized respectively with keyhole limpet hemocyanin (KLH) conjugated peptide and multiple antigen peptide (MAP). The mAb was prepared by hybridoma technique and the type of Ig was identified. The specificity of the mAb  was determined by Western blot and immunofluorescent staining. One clone of hybridoma cell secreting mAb against NMDAR1 specific B cell epitope was obtained by KLH conjugated peptide method. It belongs to IgM. Western blot analysis and immunofluorescent staining showed specific binding of mAb with corresponding antigen in brain tissue of SD rat. No positive mAb was gained from MAP immunized mice. It was found that the KLH-coupled peptide had better immunogenicity and was easier to produce the antibody against specific B cell epitope than MAP. The mAb against specific B cell epitope in NMDAR1 was obtained successfully. It is a useful tool for the functional study of NMDAR1 and for potential treatment of epilepsy, brain eschemia and so on.
Key words: NMDAR1; B cell epitope; Monoclonal antibody; Therapeutic antibody