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本试验选择肠出血性大肠杆菌O157∶H7 LEE毒力岛上的eae基因原核表达产物intimin作为抗原免疫BALB/c小鼠,经过细胞融合和4次亚克隆后获得了5株杂交瘤细胞,分别命名为1E8、2B11、3B6、4C10、5D5,效价均在1∶128 000以上。杂交瘤细胞的染色体结果显示5株杂交瘤细胞的染色体数目在90~100条之间。Western blotting鉴定结果表明所制备的单克隆抗体能特异性识别肠出血性大肠杆菌O157∶H7 intimin蛋白。间接免疫荧光显示单抗能够与肠出血性大肠杆菌O157∶H7表面抗原结合,荧光镜下可见明显绿色荧光。交叉试验结果显示制备的5株单克隆抗体均不与鸡的其它血清型大肠杆菌(O1、O2、O78、O157)及鸡白痢沙门菌等发生交叉反应,确定为肠出血性大肠杆菌O157∶H7特异性单克隆抗体。
In this study, five strains of hybridoma cells were obtained by immunization of BALB / c mice with the eti gene prokaryotic expression product intimin of enterohemorrhagic Escherichia coli O157: H7 LEE virulent island after cell fusion and 4 subclones, respectively Named as 1E8,2B11,3B6,4C10,5D5, the titer is above 1: 128 000. Chromosome results of hybridoma cells showed that the chromosome numbers of the five hybridoma cells ranged from 90 to 100. The result of Western blotting showed that the monoclonal antibody could specifically recognize enterohemorrhagic Escherichia coli O157: H7 intimin protein. Indirect immunofluorescence showed that McAb could bind to enterohemorrhagic Escherichia coli O157: H7 surface antigen, and obvious green fluorescence was observed under fluorescence microscope. The results of cross-test showed that none of the five monoclonal antibodies produced cross-reacted with other serotypes of Escherichia coli (O1, O2, O78, O157) and Salmonella pullorum, and were identified as enterohemorrhagic Escherichia coli O157: H7 Specific monoclonal antibodies.