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目的:原核表达空肠弯曲菌CjaA蛋白,并制备其单克隆抗体(mAb)。方法:克隆目的基因并将其构建到pGEX-6p-1和pET30a(+)表达载体,分别以变复性纯化后的rGST-cjaA、rHis-cjaA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mAb腹水效价,Dot-ELISA、Western blot分析mAb特异性。结果:成功构建pET30a(+)-cjaA和pGEX-6p-1-cjaA重组原核表达质粒,并融合表达rHis-cjaA和rGST-cjaA蛋白,Western blot试验显示全菌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CjaA的单克隆杂交瘤细胞株,分别命名为2B6、3C2、4F11,其Ig亚类均为IgG1,腹水效价分别为1∶1×105、1∶2×105和1∶4×105;Western blot试验显示,3株mAb均能与表达rHis-CjaA重组蛋白的细菌发生特异性反应;Dot-ELISA试验表明,3株mAb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mAb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CjaA蛋白的生物学特性、致病机制,以及建立快速检测技术奠定基础。
Objective: Prokaryotic expression of Campylobacter jejuni CjaA protein and preparation of its monoclonal antibody (mAb). METHODS: The target gene was cloned and then cloned into pGEX-6p-1 and pET30a (+) expression vector. The purified rGST-cjaA and rHis-cjaA proteins were used as immunogens filter. The ascites titer of the cell supernatant and the mAb were determined by indirect ELISA. The mAb specificity was analyzed by Dot-ELISA and Western blot. Results: The prokaryotic expression plasmids pET30a (+) - cjaA and pGEX-6p-1-cjaA were successfully constructed and fused with the rHis-cjaA and rGST-cjaA proteins. Western blot showed that the whole multi-antiserum could bind to the expressed protein in vitro The specific reaction showed that the expressed protein was immunogenic. Three strains of monoclonal anti-CjaA monoclonal cell lines secreting stable against CjaA were screened and named as 2B6, 3C2 and 4F11 respectively. The Ig subclasses were all IgG1. The ascites titers were 1: 1 × 105 and 1: 2 × 105, respectively 1: 4 × 105. Western blot showed that all 3 mAbs reacted specifically with the bacteria expressing rHis-CjaA recombinant protein. Dot-ELISA assay showed that all 3 mAbs could react with Campylobacter jejuni isolates Specific reaction occurred. Conclusion: The mAb prepared in this study has high specificity and good application value. This study laid the foundation for further study on the biological characteristics of CjaA protein, pathogenic mechanism and establishment of rapid detection technology of Campylobacter jejuni.