人肥大细胞羧基肽酶的单抗2A9识别表位的预测和鉴定医学论文

人肥大细胞羧基肽酶的单抗2A9识别表位的预测和鉴定_医学论文 作者：周艳春，侯一峰，陈霖霏，郑晓璇，何韶衡 【关键词】 肥大细胞羧基肽酶；表位,B淋巴细胞；预测；基因表达【Abstract】 AIM:

人肥大细胞羧基肽酶的单抗2A9识别表位的预测和鉴定_医学论文 作者：周 艳春，侯一峰，陈霖霏，郑晓璇，何韶衡 【关键词】 肥大细胞羧基肽酶；表位,B淋巴细胞；预测；基因表达 【Abstract】 AIM: To predict the Bcell epitope of human mast cell carboxypeptidase （hMCCP）and identify the region of Bcell epitope recognized by monoclonal antibody （mAb）against hMCCP. METHODS: The Bcell epitopes were predicted by secondary structure (SOMPA, selfoptimized prediction method with alignment), hydrophilicity （Hopp&Woods）, antigenic index (JamesonWolf) and surface probability plot (Emini). The cDNA encoding the Nterminal 1～111(P1P2), 97～ 202(P3P4) and 1～202(P1P4)Aa of hMCCP was amplified by PCR and cloned into prokaryotic expression vector pET44 Ek/LIC. After induction with IPTG, the recombinant fusion protein was expressed and analyzed by SDSPAGE. For the epitope analysis, mAb 2A9 was used to react with the fusion protein by Western blot. RESULTS: The predicted Bcell epitopes were probably located in Nterminal 1～202 Aa. mAb 2A9 could react with fusion proteins which contained P1P4 and P3P4 in Western blot analysis, but not react with fusion proteins containing P1P2. CONCLUSION: Prediction of the Bcell epitope for hMCCP can provide a base for the studies of structure and function of hMCCP. The epitope recognized by mAb 2A9 is located in 112202 Aa of hMCCP. 【Keywords】 mast cell carboxypeptidase（MCCP）, epitopes, Blymphocyte, forecasting, gene expression 【摘要】 目的： 分析人肥大细胞羧基肽酶（hMCCP）的单克隆抗体（mAb）2A9 识别表位所在区域. 方法： 根据肥大细胞hMCCP的二级结构、亲水性、表面可 能性和抗原性指数，对hMCCP的B细胞表位进行预测. 依据预测结果，采用缺 失突变的方法，分别扩增编码hMCCP N端第1～111(P1P2),97～202(P3P4)和 1～202(P1P4)位氨基酸的cDNA，并构建重组原核表达载体pET44/P1P2、 pET44/P3P4和pET44/P1P4. 用IPTG诱导表达融合蛋白，并对表达产物进行 SDSPAGE和Western blot分析. 结果： B细胞表位预测结果显示，hMCCP的N 端第1～202位氨基酸含有多个潜在的B细胞表位. PCR扩增出的3个目的片 段，其DNA序列同NCBI公布的序列完全一致. Western blot结果显示： E.coli BL21 (DE3)表达的融合蛋白P3P4和P1P4均与mAb 2A9反应；而融合蛋 白P1P2与mAb 2A9不发生反应. 结论： 用多参数综合测评的方法，可获得满 意的hMCCP B细胞表位预测结果，本研究中mAb 2A9识别的表位位于hMCCP的 112202位氨基酸区域. 【关键词】 肥大细胞羧基肽酶；表位，B淋巴细胞；预测；基因表达 人肥大细胞羧基肽酶(human mast cell carboxypeptidase, hMCCP)是一种存在 于结缔组织肥大细胞内的中性蛋白酶，约占肥大细胞蛋白总量的12%. 成熟的