噬菌体肽库中筛选NMDA受体抗原表位  

Screening of a NMDA Receptor Epitope From Random Phage Display Peptide Library

作　　者：康晓楠[1] 孙长凯[1] 范明[2] 薛沿宁[2] 邵宁生[2] 赵杰[1] 韩大跃[3] 施广霞[4] 

机构地区：[1]大连医科大学脑疾病研究所,大连116027 [2]军事医学科学院基础医学研究所,北京100850 [3]解放军第210医院,大连116021 [4]大连医科大学病理生理学教研室,大连116027

出　　处：《生物化学与生物物理进展》2003年第4期599-604,共6页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目 (3 0 0 70 2 67);中国博士后科学基金 (2 0 0 1);辽宁省重点科技攻关课题项目 (2 0 0 12 2 60 0 5 )~~

摘　　要：为了获得人N 甲基 D 天冬氨酸受体 (NR)主亚基 (NR1)M3 M4环B细胞表位 ,以人NR1分子M3 M4环单克隆抗体MAB36 3淘筛噬菌体展示随机 12肽库 ,对筛选克隆进行特异性ELISA检测和竞争结合实验分析 .从 30个克隆中得到一个阳性克隆序列“VHTNPSTWQPIL” (克隆 1) ,原核表达的NR1M 3 M 4环可以与克隆 1竞争结合MAB36 3.固相合成 5个表位探针短肽 ,发现其中只有R (2 2 )LRNPSKD可以与M3 M4环竞争结合抗体 ,将NPS三个氨基酸残基逐一敲除 ,缺失N或NP的合成肽和M 3 M 4环竞争抗体的能力减弱 ,缺失NPS的合成肽完全没有竞争能力 ,证明MAB36 3的表位为NPS ,S可能是关键性残基 .To determine the B cell epitope of a monoclonal antibody against the M3-M4 loop of NMDAR1, a random phage displayed dodecapeptide library was screened with the monoclonal antibody MAB363 against the M3-M4 loop of NMDAR1. After four rounds of biopanning, the peptide sequences of positive phage clones were determined and analyzed by DNA sequencing, ELISA and competitive inhibition assay. A positive clone was found (clonel: VHTNPSTWQPIL). The binding between clonel and MAB363 were competitively inhibited by the recombined M3-M4 loop expressed by E. coli DH5alpha; The binding between M3M4 and MAB363 could be competitively inhibited by one of solid-synthesized epitope peptides: RLRNPSKD. There were identical sequences among them: NPS. Deleted with NPS, the peptide could not inhibit the binding of MAB363 to M3-M4. These results demonstrate that NPS in M3-M4 loop is the B cell epitope recognized by MAB363, which may be important for developing a practical immunization strategy against excitotoxic brain injury.

关 键 词：噬菌体 肽库 NMDA受体 抗原表位 N-甲基-D天冬氨酸受体 

分 类 号：Q939.48[生物学—微生物学] Q26