空肠弯曲菌cjaA基因表达蛋白单克隆抗体的制备及其生物学特性鉴定

Preparation and characterization of monoclonal antibodies specific for CjaA protein of Campylobacter jejuni

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。

AIM： Expression, purification of Campy- Iobacter jejuni CjaA protein and development of monoclonal antibodies （mAbs） against this protein. METHODS： The C. jejuni cjaA gene was amplified and inserted into the expression plasmids, pGEX-6p-1 and pET30a （ ＋ ）. The purified rGST-CjaA protein was used as an immunogen in 8- week-old BALB/c mice, and injected subcutaneously. The purified rHis-CjaA protein used as a detecting antigen for screening mAbs against CjaA was prepared. The specificity of mAbs was characterized by Dot-ELISA and Western blot assays. RESULTS： The recombinant expression plasmids, pGEX-6p-l-cjaA and pET30a（ ＋ ）-cjaA were obtained. The sizes of the recombinant proteins, rGST-CjaA and rHis- CjaA, were consistent with their predicted size. Specific reaction was found between CjaA positive serum and expressed protein by Western blot assay, confirming its identification as a Campylobacter jejuni immunogen. Three hybridoma cell lines, designated 2B6, 3C2 and 4Fll, secreting mAbs against CjaA were obtained. Their immunoglobulin subclasses were all IgGl. The ELISA titers of the ascites fluid were 1：1× 10^5, 1：2 × 10^5 and 1：4× 10^5 , respectively. Western blot analysis confirmed that the three mAbs reacted with the rHis-CjaA fusion protein but not the His tag. The Dot-ELISA results demonstrated that the three mAbs only with CjaA and not the tags for the expression vectors. CONCLUSION： The successful preparation of three mAbs specific for the CjaA protein lays the foundation for further study regarding the biological characteristics of CjaA and the pathogenesis of C. jejuni.