抗人α-烯醇化酶单克隆抗体的研制及其抗原识别表位初步鉴定

Development and epitope mapping of a monoclonal antibody against humanα-enolase

研制抗人α-烯醇化酶的单克隆抗体,并对其生物学特性进行初步研究。利用原核表达系统对人α-烯醇化酶进行一系列重组表达。表达纯化后的人α-烯醇化酶作为抗原免疫BALB/c小鼠,采用杂交瘤技术以及克隆化培养筛选稳定分泌抗人α-烯醇化酶单克隆抗体的杂交瘤细胞株,采用免疫荧光技术、免疫组化技术对此单克隆抗体加以验证,并应用噬菌体随机十二肽库展示技术结合生物信息学方法对抗体识别表位进行初步鉴定。结果显示,克隆表达了人α-烯醇化酶,筛选获得一株稳定分泌抗人α-烯醇化酶单克隆抗体细胞株,此单克隆抗体的亚型为IgG1,它可与腹腔巨噬细胞的胞膜及胞浆的烯醇化酶特异性结合。噬菌体随机十二肽库展示技术筛选出的氨基酸序列经生物信息学分析,初步确定此单克隆抗体的抗原表位为第259-262氨基酸与第267-269氨基酸,其恰好位于人α-烯醇化酶三维结构表面的凸环上。成功制备了一株抗人α-烯醇化酶单克隆抗体并对其抗原识别表位进行初步鉴定,为探究人α-烯醇化酶在人体病理生理活性中的作用提供了研究工具。

We set out to generate monoclonal antibodies against human α-enolase and identify the antigen-specific epitope. The recombinant human ccenolase protein was expressed by the prokaryotic system, and monoclonal antibodies against human α-enolase were generated. Epitopes of the mAb were analyzed by phage display peptide library. The specificity of the mAb was investigated by indirected ELISA, immunofluorescence （IF） and immunohistochemistry （IHC）. The results showed that human α-enolase was expressed successfully. A mAb against human α-enolase was produced , which was of IgG1 subtype. Immunofluorescence test and immunohistochemistry test conformed that the mAb could recognize the enolase in murine microphages. Epitopes of the mAb were mapped through phage display peptide library and bioinformatics. Altogether, a mAb against human α-enolase is generated which may supply an effective tool to analyze the mechanism of human α-enolase in human pathological and physiological activities.