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【目的】原核表达空肠弯曲菌鞭毛蛋白FlaA,并制备其单克隆抗体。【方法】克隆目的基因并将其构建到pET30a(+)和pGEX-6p-1表达载体,分别以变复性纯化后的rHis-FlaA、rGST-FlaA蛋白为免疫原和检测原进行杂交瘤细胞的筛选。采用间接ELISA法测定细胞上清和单抗腹水效价,Dot-ELISA、Western blot分析单抗特异性。【结果】成功构建pET30a(+)-flaA和pGEX-6p-1-flaA重组原核表达质粒,并融合表达rHis-FlaA和rGST-FlaA蛋白,Western blot试验显示天然蛋白多抗血清能与体外表达的蛋白呈现特异性反应,表明表达蛋白具有免疫原性。筛选获得3株稳定分泌抗FlaA的单克隆杂交瘤细胞株,分别命名为2D12、5E12、6A9,其Ig亚类分别为IgG2a、IgG1、IgG1,腹水效价分别为1∶102400,1∶102400和1∶51200;Western blot试验显示,3株单抗均能与表达rHis-FlaA重组蛋白的细菌发生特异性反应;Dot-ELISA试验表明,3株单抗均能与不同来源的空肠弯曲菌分离株发生特异性反应。【结论】本研究制备的单克隆抗体有较高特异性,具有良好的应用价值。为进一步研究空肠弯曲菌鞭毛蛋白的生物学特性、致病机理,以及建立快速检测技术奠定基础。
【Objective】 Prokaryotic expression of Campylobacter jejuni flaA FlaA and preparation of its monoclonal antibody. 【Method】 The target gene was cloned and cloned into pET30a (+) and pGEX-6p-1 expression vector. The purified rHis-FlaA and rGST-FlaA protein were used as immunogen and test antigen respectively for hybridoma cells Screening. The ascites titer of the cell supernatant and the monoclonal antibody was measured by indirect ELISA, and the specificity of the monoclonal antibody was analyzed by Dot-ELISA and Western blot. 【Results】 The prokaryotic expression plasmids pET30a (+) - flaA and pGEX-6p-1-flaA were successfully constructed and fused with the rHis-FlaA and rGST-FlaA proteins. Western blot showed that the native polyclonal antiserum could be expressed in vitro Protein showed specific reaction, indicating that the expressed protein is immunogenic. Three monoclonal anti-FlaA secreting McAbs were obtained and named as 2D12, 5E12 and 6A9, respectively. The Ig subtypes were IgG2a, IgG1 and IgG1, the ascites titers were 1:102400, 1:102400 and 1: 51200; Western blot test showed that all three McAbs reacted specifically with the bacteria expressing rHis-FlaA recombinant protein. Dot-ELISA showed that all three McAbs could react with Campylobacter jejuni isolates Specific reaction occurred. 【Conclusion】 The monoclonal antibodies prepared in this study have high specificity and good application value. It laid the foundation for further study of the biological characteristics of Campylobacter jejuni flagellin, pathogenesis and establishment of rapid detection technology.