大肠杆菌O157：H7外膜蛋白C重组表达及免疫原性研究

Recombinant Expression and Immunogenicity of the Outer Membrane Protein C of Escherichia coli O157:H7

摘要:

目的 探究大肠杆菌（Escherichia coli， E. coli） O157：H7外膜蛋白C （OmpC）的免疫原性。方法 分别构建pET-28a-ompc、pET-22b-ompc-SUMO重组质粒，表达纯化重组蛋白OmpC，并验证大肠杆菌O157：H7菌体免疫制备的2G12抗体的识别抗原是否为OmpC。结果 作者成功构建了2种重组质粒，其中pET-28a-ompc在E. coli BL21中成功表达；重组蛋白OmpC的最佳诱导表达条件为：0.2 mmol/L IPTG、37 ℃诱导12 h；在800 mmol/L咪唑洗脱时得到含有His标签的包涵体OmpC，通过尿素梯度复性结合Ni-NTA柱纯化，在500 mmol/L咪唑洗脱时获得可溶性OmpC。结论 可溶性OmpC和包涵体OmpC均能与单克隆抗体2G12发生特异性反应。该研究为后续利用OmpC制备高亲和力的大肠杆菌O157：H7单克隆抗体奠定了基础。

Abstract:

Objective This study aims to investigate the immunogenicity of the outer membrane protein C (OmpC) of Escherichia coli (E. coli) O157:H7.Method The recombinant plasmids pET-28a-ompc and pET-22b-ompc-SUMO were constructed, and the recombinant protein OmpC was expressed and purified. Then, whether the target antigen of the 2G12 antibody prepared by E. coli O157: H7 cells was OmpC was determined.Result Two recombinant plasmids were successfully constructed and pET-28a-ompc was successfully expressed in E. coli BL21. The optimal induction conditions for expression of recombinant OmpC were 0.2 mmol/L IPTG, 37 ℃, and 12 h. The OmpC inclusion bodies containing His tags were obtained through elution with 800 mmol/L imidazole. The soluble OmpC was renatured by urea gradient dialysis and then eluted from the Ni-NTA column with 500 mmol/L imidazole.Conclusion OmpC in both the soluble form and the inclusion body form specifically react with the monoclonal antibody 2G12. The findings lay a foundation for the further preparation of high-affinity E. coli O157:H7 monoclonal antibodies with OmpC.