重组创伤弧菌溶细胞素单克隆抗体的制备

目的：制备重组创伤弧菌溶细胞素（recombinant Vibrio vulnificus cytolysin,rVVC）鼠源性单克隆抗体,并对其特异性及免疫球蛋白类型进行鉴定,为创伤弧菌溶细胞素（Vibrio vulnificus cytolysin,VVC）致病机制的后续研究及创伤弧菌引起的食品污染中溶细胞毒素快速检测试剂盒的研发奠定基础。方法：IPTG诱导含pET28a（＋）-vvhA的大肠杆菌表达rVVC,将rVVC纯化、复性后经甲醛脱毒作为抗原免疫BALB/c小鼠。SDS-PAGE检测蛋白纯化后效果。采用杂交瘤技术和ELISA法制备并筛选分泌rVVC单克隆抗体的杂交瘤细胞株,有限稀释法对阳性孔克隆化培养。采用免疫双扩法鉴定单克隆抗体类型,ELISA法、免疫双扩法鉴定单克隆抗体的特异性。结果：将纯化复性后的rVVC作为抗原免疫小鼠后,经ELISA检测,血清有效稀释度达1：12800。共获得2株可持续分泌单克隆抗体的杂交瘤细胞株,分别为B2C7和E3F4株,其抗体类型均为IgG1,且具有较高特异性,与多种其他细菌蛋白不发生免疫反应。结论：本研究成功免疫BALB/c小鼠,获得B2C7和E3F4两株可持续稳定分泌rVVC鼠源性单克隆抗体的杂交瘤细胞株,抗体类型为IgG1型。

Preparation of monoclonal antibody against recombined cytolysin of Vibrio vulnificus

Objective： Mouse-original monoclonal antibody against recombinant Vibrio vulnificus cytolysin （rVVC） was prepared and its specificity and type of immunoglobulin were identified,which can lay a foundation for the further study of Vibrio vulnificus cytolysin （VVC） pathogenesis and the development of rapid detection kit of Vibrio vulnificus cytolysin.Methods： IPTG was used to induce E.coli BL21 （DE3） pET28a（＋）-vvhA to express rVVC.The rVVC which purificated,refolded and then detoxified was used to immune BALB/c.The effects of expression and purification of rVVC were determined by SDS-PAGE.The hybridoma cell lines to secrete anti-rVVC monoclonal antibodies were prepared and screened by hybridoma technique and ELISA,and then were cloned with limited dilution.Double immunodiffusion assay was used to identify the type and subclass of monoclonal antibody.ELISA and double immunodiffusion assay were used to determine the specificity.Results： The mice immunized by rVVC which had been purified,refolded and detoxified showed the highest serum dilution to 1：12 800 by ELISA.Two cell lines of hybridoma were screened out,and cells of B2C7 and E3F4 were found to be continuously secreting IgG1 monoclonal antibodies.The monoclonal antibodies showed high specificity,and didn’t react with the proteins from other bacteria.Conclusion： BALB/c mice is successfully immunized and two mouse-origin hybridoma cell lines named B2C7 and E3F4 which continually and steadily secreted specific monoclonal antibodies against rVVC are acquired.The type of monoclonal antibody against rVVC is IgG1.