人肥大细胞羧基肽酶的单抗2A9识别表位的预测和鉴定  

Prediction of B cell epitope of human mast cell carboxypeptidase and its identification by monoclonal antibody 2A9

作　　者：周艳春[1] 侯一峰[1] 陈霖霏[1] 郑晓璇[1] 何韶衡[1] 

机构地区：[1]汕头大学医学院变态反应与炎症学研究所,广东汕头515041

出　　处：《第四军医大学学报》2006年第20期1863-1866,共4页Journal of the Fourth Military Medical University

基　　金：广东省自然科学基金重点项目(04106122)

摘　　要：目的：分析人肥大细胞羧基肽酶（hMC-CP）的单克隆抗体（mAb）2A9识别表位所在区域．方法：根据肥大细胞hMC-CP的二级结构、亲水性、表面可能性和抗原性指数，对hMC-CP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hMC-CPN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pET-44／P1P2、pET-44／P3P4和pET-44／P1P4．用IPTG诱导表达融合蛋白，并对表达产物进行SDS-PAGE和Western blot分析．结果：B细胞表位预测结果显示，hMC-CP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 BI21（DE3）表达的融合蛋白P3P4和P1P4均与mAb2A9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hMC-CPB细胞表位预测结果，本研究中mAb 2A9识别的表位位于hMC-CP的112-202位氨基酸区域．AIM： To predict the B cell epitope of human mast cell carboxypeptidase （hMC-CP）and identify the region of B cell epitope recognized by monoclonal antibody （ mAb ） against hMC-CP. METHODS： The B cell epitopes were predicted by secondary structure （SOMPA, self-optimized prediction method with alignment）, hydrophilicity （Hopp＆Woods） , antigenic index （Jameson-Wolf） and surface probability plot （Emini）. The cDNA encoding the N terminal 1 - 111 （ P1 P2 ）, 97 - 202 （ P3 P4 ） and 1 - 202 （ P1 P4 ） Aa of hMC-CP was amplified by PCR and cloned into prokaryotic expression vector pET-44 Ek/LIC. After induction with IPTG, the recombinant fusion protein was expressed and analyzed by SDS-PAGE. For the epitope analysis, mAb 2A9 was used to react with the fusion protein by Western blot. RESULTS： The predicted B cell epitopes were probably located in N terminal 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 B cell epitope for hMC-CP can provide a base for the studies of structure and function of hMC-CP. The epitope recognized by mAb 2A9 is located in 112-202 Aa of hMC-CP.

关 键 词：肥大细胞羧基肽酶 表位 B淋巴细胞 预测 基因表达 

分 类 号：R392.11[医药卫生—免疫学]