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【目的】原核表达空肠弯曲菌细胞致死性肿胀毒素B蛋白(CdtB),制备其单克隆抗体(mAb),并研究mAb抗毒性作用。【方法】扩增空肠弯曲菌cdtB基因并将其构建到pET-30a(+)和pGEX-6p-1表达载体,以原核表达的GST-CdtB蛋白为免疫原,应用杂交瘤技术进行细胞融合;采用间接ELISA方法测定细胞上清和mAb腹水效价,Dot-ELISA、Western blot分析mAb特异性,并以CaCo-2和HD-11细胞为模型,鉴定mAb抗毒性能力。【结果】成功构建重组原核表达质粒pET-30a(+)-cdtB和pGEX-6p-1-cdtB,并融合表达rHis-CdtB和rGST-CdtB蛋白。获得5株稳定分泌CdtB抗体的杂交瘤细胞株,分别命名为1F3,1F5,2E4,2E11,2F2。抗体Ig类和亚类检测显示2E11 Ig亚类为IgG2b,其他4株均为IgG1。抗体效价高达1:(1×108)。Dot-ELISA试验表明5株mAb均能与空肠弯曲菌标准株发生特异性反应,与非空肠弯曲菌呈阴性反应;Western blot法分析表明5株mAb均能与纯化蛋白rGST-CdtB有良好的反应性。基于CaCo-2细胞的黏附和侵袭实验表明mAb能显著降低细菌的黏附和侵袭能力(P<0.01)。【结论】成功制备了针对空肠弯曲菌CdtB蛋白的mAb。为进一步研究空肠弯曲菌致病机制,以及为研制治疗性类药物奠定了基础。
【Objective】 Prokaryotic expression of Campylobacter jejuni cell lethality toxin B protein (CdtB), preparation of its monoclonal antibody (mAb), and study the anti-toxicity of mAb. 【Method】 The cDNA of Campylobacter jejuni cdtB was amplified and inserted into pET-30a (+) and pGEX-6p-1 expression vector. The prokaryotic expression of GST-CdtB protein was used as the immunogen. Indirect ELISA was used to determine the titer of ascites and mAb ascites, Dot-ELISA and Western blot were used to analyze mAb specificity. The anti-toxicity ability of mAb was identified by using CaCo-2 and HD-11 cells as models. 【Results】 The recombinant prokaryotic expression plasmids pET-30a (+) - cdtB and pGEX-6p-1-cdtB were successfully constructed and fused with rHis-CdtB and rGST-CdtB proteins. Five strains of hybridoma cell lines stably secreting CdtB were obtained and designated as 1F3, 1F5, 2E4, 2E11 and 2F2, respectively. Antibody Ig class and subclass tests showed 2E11 Ig subclass IgG2b, the other four were IgG1. Antibody titers up to 1: (1 × 108). Dot-ELISA assay showed that all 5 mAbs reacted specifically with Campylobacter jejuni standard strains and negative with Campylobacter jejuni. Western blot analysis showed that all 5 mAbs reacted well with the purified protein rGST-CdtB Sex. Adhesion and invasion assays based on CaCo-2 cells demonstrated that mAb significantly reduced bacterial adhesion and invasion (P <0.01). 【Conclusion】 The mAb against Campylobacter jejuni CdtB was successfully prepared. In order to further study the pathogenic mechanism of Campylobacter jejuni, as well as the development of therapeutic drugs laid the foundation.