【摘要】 目的从噬菌体展示的随机十二肽库中筛选N-甲基-D-天门冬氨酸受体(NMDAR)2B亚基模拟抗原表位。方法以NMDAR2B单克隆抗体为配基,免疫亲和筛选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机十二肽。经过3轮筛选,从第3轮洗脱物中随机挑选12个单克隆噬菌体扩增后进行ELISA鉴定,用酶标仪测定450 nm处的吸光值(A450)。对这12个单克隆噬菌体分别进行扩增、纯化,并对DNA测序,以确定插入十二肽的氨基酸序列。通过细胞竞争ELISA法分析阳性单克隆噬菌体对NMDAR2B天然抗原表位与其特异性抗体结合的竞争性抑制。结果经过3轮筛选后能与NMDAR2B单克隆抗体特异性结合的噬菌体得到了有效富积,12个单克隆噬菌体中有9个单克隆噬菌体的A450高于其它3个。DNA测序结果表明这9个单克隆噬菌体表达了一个共同氨基酸序列:SHPPVMPWPTST,将其命名为阳性克隆噬菌体,该阳性克隆噬菌体可以竞争性抑制细胞表面天然抗原与特异性抗体的结合,抑制率(45±3)％,具有抗原模拟性。结论从噬菌体展示的随机十二肽库中成功筛选到了能与NMDAB2B特异性抗体结合的短肽,该肽模拟了天然抗原的某个表位。更多还原

【Abstract】 Objective To determine the mimic epitope of N-methyl-D-aspartate receptor 2B subunit (NR2B). Methods The monoclonal antibody interacting with NR2B was used as target protein to screen the binding peptide from a 12-mer Ph. D random peptide library. After three rounds of affinity screening, twelve specific clones were selected randomly and identified by sandwich ELISA. The peptide sequences were analyzed by DNA sequencing. The clones containing a common sequence were named positive clone. The competitive inhibition of the native antigen bound to monoclonal antibody against the NR2B by the positive clone was assayed by cell ELISA. Results After 3 rounds of screening the phages specifically bound with mAb NR2B were selected and amplified. Nine of the 12 clones displayed a common aminoacid sequence: SHPPVMPWPTST. The inhibitory assay showed that the mimic epitope peptides displayed on the phage surface could effectively inhibit the combination of the monoclonal antibody with native antigen. The inhibitory rate of mimic epitope was (45±3)% .Conclusion The mimic epitope of NR2B was screened successfully from the 12-mer Ph D random peptide library. This peptide mimics an epitope of the native NR2B.更多还原