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目的构建肠出血性大肠杆菌(EHEC)O157∶H7毒力蛋白nleB1基因的原核表达载体,诱导表达重组蛋白,制备NleB1的抗血清。方法从EHEC O157∶H7 EDL933株的全基因组中PCR扩增出编码nleB1的990个碱基序列,构建原核表达载体pET24a-nleB1,将构建的重组表达载体的质粒pET24a-nleB1转化至大肠杆菌BL21(DE3)感受态细胞中,利用异丙硫半乳糖苷(IPTG)诱导融合蛋白His-NleB1表达,并利用镍柱进行亲和层析纯化和切胶纯化,以纯化后的融合蛋白His-NleB1为抗原免疫BALB/c小鼠,获得抗血清。Western印迹和ELISA法鉴定获得的抗血清。结果成功构建了pET24a-nleB1原核表达载体,纯化得到融合蛋白His-NleB1,进而免疫BALB/c小鼠制备多克隆抗体,Western印迹和ELISA法证实多克隆抗体制备成功。结论成功获得了EHEC O157∶H7的毒力蛋白NleB1的多克隆抗体,为研究EHEC O157∶H7毒力蛋白NleB1的功能奠定了基础。
Objective To construct the prokaryotic expression vector of nleB1 gene of enterohemorrhagic Escherichia coli (EHEC) O157: H7 virulence protein and induce the expression of recombinant protein to prepare the antiserum of NleB1. Methods The 990 base sequence of nleB1 was amplified by PCR from the whole genome of EHEC O157: H7 EDL933 strain. The prokaryotic expression vector pET24a - nleB1 was constructed. The recombinant plasmid pET24a - nleB1 was transformed into E. coli BL21 DE3) competent cells, the fusion protein His-NleB1 was induced by isopropylthiogalactoside (IPTG) and purified by affinity chromatography and gel excision using a nickel column. The purified fusion protein His-NleB1 was BALB / c mice were immunized with antigens to obtain antiserum. The obtained antiserum was identified by Western blotting and ELISA. Results The prokaryotic expression vector pET24a-nleB1 was successfully constructed, and the fusion protein His-NleB1 was purified. BALB / c mice were immunized to prepare polyclonal antibodies. Western blotting and ELISA confirmed the successful preparation of polyclonal antibodies. Conclusion The polyclonal antibody against virulence protein NleB1 of EHEC O157:H7 was successfully obtained, which laid the foundation for the study of the function of EHEC O157:H7 virulence protein NleB1.