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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。
OBJECTIVE: To prepare a murine monoclonal antibody against recombinant Vibrio vulnificus cytolysin (rVVC), and to identify its specificity and immunoglobulin types. The aim of this study was to investigate the effects of Vibrio vulnificus cytolysin VVC) pathogenic mechanism of the follow-up study and Vibrio vulnificus caused by food contamination in cytotoxic test kit rapid basis for the research and development. Methods: The E. coli expressing pET28a (+) - vvhA was induced by IPTG to express rVVC. The rVVC was purified and renatured. After renaturation, BALB / c mice were immunized with formaldehyde as an antigen. SDS-PAGE test protein purification effect. Hybridoma cell lines secreting rVVC monoclonal antibody were prepared and screened by hybridoma technology and ELISA, and positive clones were cultured by limited dilution method. Monoclonal antibody types were identified by double immunodiffusion method, and the specificity of monoclonal antibodies was identified by ELISA and double immunodiffusion. Results: After purification and purification of rVVC as antigen immunized mice, the serum was effectively diluted to 1: 12800 by ELISA. A total of 2 hybridoma cell lines secreting monoclonal antibodies were obtained. They were B2C7 and E3F4 strains, all of which were of IgG1 antibody type with high specificity and did not immunoreact with a variety of other bacterial proteins. CONCLUSION: In this study, BALB / c mice were successfully immunized with two strains of hybridoma cell line B2C7 and E3F4 that can stably secrete rVVC murine monoclonal antibody. The antibody type was IgG1.