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目的:利用大肠杆菌重组表达金黄色葡萄球菌外分泌蛋白Efb(extracellular fibrinogen-binding protein,细胞外纤维蛋白原结合蛋白),检测其生物学活性,并制备其功能性抗体,以探讨Efb蛋白在金葡菌感染中的生物学意义。方法:以金黄色葡萄球菌NCTC-8325菌株基因组为模板,PCR扩增efb基因,构建重组表达载体pET28a-Efb,转化大肠杆菌BL21(DE3),利用IPTG诱导表达,通过Ni柱亲和纯化获得Efb;通过补体激活试验及竞争ELISA的方法检测Efb蛋白的生物学活性,免疫动物制备抗体。结果:获得了纯度较高的重组Efb蛋白,重组蛋白能够有效抑制CH50以及AH50,并且制备了Efb的功能性多克隆抗体。结论:Efb蛋白能够抑制补体激活的经典途径及替代途径,其特异性抗体能够阻断对于补体激活经典途径的抑制作用。
OBJECTIVE: To detect the biological activity of Escherichia coli by recombinantly expressing extracellular fibrinogen-binding protein (Efb) and to prepare its functional antibody to explore the role of Efb protein The biological significance of bacterial infection. Methods: The efb gene was amplified by PCR using the genome of Staphylococcus aureus NCTC-8325 as a template. The recombinant expression vector pET28a-Efb was constructed and transformed into E. coli BL21 (DE3). The recombinant plasmid was induced by IPTG and purified by Ni column affinity purification. The biological activity of Efb protein was detected by complement activation assay and competitive ELISA, and the animals were immunized to prepare antibodies. Results: The recombinant Efb protein with high purity was obtained. The recombinant protein could effectively inhibit CH50 and AH50, and the functional polyclonal antibody of Efb was prepared. CONCLUSION: Efb protein can inhibit the classical pathway and alternative pathway of complement activation, and its specific antibodies can block the inhibitory effect on the classical pathway of complement activation.