创伤弧菌溶细胞毒素单克隆抗体制备及其鉴定

目的制备创伤弧菌(Vibrio vulnificus)溶细胞素vvhA基因产物鼠源性单克隆抗体并鉴定其特异性和免疫性,为进一步研制创伤弧菌检测试剂盒奠定基础。方法采用IPTG诱导目的重组蛋白rvvhA表达,Ni-NTA亲和层析法提纯rvvhA,SDS-PAGE检测表达和提纯效果。采用杂交瘤技术和rvvhA-ELISA制备并筛选分泌rvvhA单克隆抗体的细胞株,有限稀释法进行细胞克隆。采用免疫双扩散法鉴定单克隆抗体类型。采用ELISA、免疫双扩散法和Western Blot鉴定单克隆抗体的效价和特异性。结果在0.5mmol/L IPTG诱导下,rvvhA产量可占细菌总蛋白的18%。提纯的rvvhA经SDS-PAGE后仅显示单一的蛋白条带。共获得9株rvvhA抗体阳性的杂交瘤细胞株,其中A5E8和C3B6株可持续分泌高效价特异性单克隆抗体,其抗体类型分别为IgG1和IgG2a。A5E8和C3B6单克隆抗体有较高特异性,与多种其它细菌蛋白不发生免疫反应,对rvvhA及创伤弧菌GTC333株和WZ01株蛋白的ELISA检测阳性的效价可达1∶4000~1∶8000、免疫双扩散效价为1∶4~1∶8,Western Blot结果显示此等单克隆抗体均能有效识别rvvhA。结论本研究成功地获得了2株稳定分泌rvvhA特异性单克隆抗体的鼠源性杂交瘤细胞株,rvvhA单克隆抗体可用于检测自然表达的创伤弧菌溶细胞素。

Preparation and identification of monoclonal antibody against cytolysin of Vibrio vulnificus

To prepare mouse-original monoclon antibody against the product of vvhA gene encoding Vibrio vulnificus cytolysin and to identify its specificity and immunogenicity in order to lay a foundation for develment of a diagnostic kit to detect V. vulnificus,IPTG was used to induce the target recombinant protein rvvhA and Ni-NTA affinity chromatography was applied to extract rvvhA. By using SDS-PAGE plus BioRad Agarose Image Analysor, the effects of expression and purification of rvvhA were determined. The cell lines to secrete anti-rvvhA monoclonal antibodies were prepared and screened by hybridoma technique and rvvhA-ELISA, and then were cloned by limited dilution method. ELISA, immunodiffusion assay and Western blot hybridization were used to tetect the titers and specificity.Under induction with 0.5 mmol/L IPTG, the output of rvvhA could reach 18% of total bacterial proteins. The purified rvvhA only showed a single fragment in gel after SDS-PAGE. Nine cell lines of hybridoma were screened out, and lines A5E8 and C3B6 were found to be continuously secreting specific IgG1 or IgG2a monoclonal antibodies with high titers, respectively. This monoclonal antibodies showed high specificity,not reacting with the proteins from other bacteria.The titers in ELISA and immunodiffusion to rvvhA and the proteins from Vibrio vulnificus strains GTC333 and WZ01 were as high as 1∶4000∶1∶8000 and 1∶4-1∶8, respectively. The result of Western blot hybridization indicated that the monoclonal antibodies could recognize rvvhA efficiently. Two mouse-origin cell lines of hybridoma stably secreting specific monoclonal antibodies against rvvhA were obtained in this study. This anti-rvvhA monoclonal antibodies could be used to detect naturally secreted cytolysin of Vibrio vulnificus.