COMP特异性单抗15A11的表位特征研究

Identification of epitope recognized by m Ab 15A11 sepecific against cartilage oligomeric matrix protein

目的:鉴定RA相关自身抗原COMP的一株特异性单克隆抗体15A11的表位特征。方法:选用随机十二肽噬菌体库对m Ab 15A11进行三轮筛选,随机挑取40个单噬菌斑,提取DNA,测序;ELISA检测每个噬菌体克隆与m Ab 15A11结合的特异性;通过Clustal W2对特异性结合的噬菌体展示的十二肽和COMP进行氨基酸序列比对,Py MOL分析一致氨基酸所在肽链的二级结构及表位氨基酸之间的距离,初步确定m Ab 15A11的表位;变性和非变性Western blot、EDTA螯合Ca2+后ELISA分析以及合成多肽的ELISA实验进一步确定表位序列。结果:得到的40个测序噬菌体中,共有5个噬菌体克隆,ELISA确定克隆1和克隆2与m Ab 15A11特异性结合,其他克隆均为非特异性结合的噬菌体;氨基酸序列比对,在COMP上未发现与克隆1和克隆2噬菌体相同的连续氨基酸序列,提示m Ab 15A11的抗原表位可能为非线性表位;Py MOL分析表位氨基酸在COMP上的定位及距离,显示构象表位的合理性;变性Western blot分析为阴性,而非变性Western blot条件下为阳性;EDTA螯合Ca2+破坏COMP的构象后不能与m Ab 15A11结合,而未经处理的与m Ab 15A11结合,均说明m Ab 15A11表位是构象表位;合成多肽与m Ab 15A11的ELISA结果进一步确定了m Ab 15A11的构象表位序列。结论:鉴定了m Ab 15A11的表位是构象表位序列,且确定了该构象表位的氨基酸组成,为研究COMP抗体与抗原反应机制具有重要理论价值,并对类风湿性关节炎的检测有重要应用意义。

Objective： To identify the epitope of mAb15A11 which is specific against RA associated autoantigen cartilage oligomeric matrix protein（ COMP）. Methods： A filamentous phage library displaying random linear dodecapeptides was used to mapping the epitope of mAb15A11. After three rounds of screenings,40 phage clones were selected at random and sequenced. The specificity of phages was confirmed by enzyme immunoassays. Homology search by Clustal W2 and structure analysis by Py Mol to identified the epitope amino acid sequence. Western blot analysis of COMP and ELISA analysis of COMP-derived peptides were used to confirm epitope＇s characterization. Results： After repeated screenings using bio-panning method,2 clones were identified,which interacted specifically with mAb 15A11. Homology search did not find succession consensus sequence within COMP molecular,which indicated that the epitope was not linear. Py Mol Structure analysis identified the rationality of conformational epitope. Western blot analysis and ELISA of EDTA-treated COMP further prove an conformational structure of the epitope recognized by mAb 15A11. ELISA analysis of COMP-derived peptides demonstrated both disulfide bonds between229C-243 C and237C-253 C and every epitope amino acid of232 G,238H,240 H,241A,244 V,247R and251 R were essential to the binding of mAb 15A11 with COMP. Conclusion： In this study,the potential B cell antigentic epitopes of mAb15A11 was identified by phage display library. The epitope amino acids sequence and characterization were also recognized. It may have important theoretical value for the study of reaction mechanism of COMP antibody and antigen and may also show application significance in the detection of rheumatoid arthritis.