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目的利用大肠杆菌重组表达金黄色葡萄球菌(SAU)重要的外分泌毒力因子α-溶血素(alpha hemolysin,Hla),检测Hla蛋白对不同种属红细胞的裂解作用;制备其特异性抗体,并检测抗体的中和活性。方法以SAU NCTC-8325菌株基因组为模板,PCR扩增hla基因,构建重组表达载体p ET-28a-Hla,转化大肠杆菌BL21(DE3),利用IPTG诱导表达,通过Ni2+柱亲和纯化获得Hla蛋白;通过红细胞裂解实验测定Hla蛋白的溶血活性。结果重组Hla蛋白以可溶形式表达,细胞裂解实验表明,纯化的Hla蛋白可以显著裂解兔红细胞,但人红细胞和羊红细胞对Hla杀伤作用不敏感。ELISA结果表明,获得了效价较高的多克隆抗体。该抗体可以有效抑制Hla对兔红细胞的裂解作用。结论 SAU的Hla蛋白对不同种属红细胞的溶解能力不同,为探讨Hla裂解细胞的机制给予一定的提示;同时所制备的特异性抗体(anti-Hla)可以抑制Hla的溶血作用,为深入研究Hla的致病机制奠定了基础。
OBJECTIVE: To detect the cleavage effect of Hla protein on erythrocytes of different species using Escherichia coli recombinant expression of the important exocrine virulence factor α-hemolysin (Hla) of S. aureus (SAU); to prepare its specific antibodies and to detect Antibody neutralizing activity. Methods The hla gene was amplified by PCR from the genome of SAU NCTC-8325 strain. The recombinant plasmid p ET-28a-Hla was constructed and transformed into E. coli BL21 (DE3). The recombinant plasmid was induced by IPTG and purified by Ni2 + affinity chromatography. Hemolysis activity of Hla protein was determined by erythrocyte lysis assay. Results The recombinant Hla protein was expressed in soluble form. Cell lysis assay showed that purified Hla protein could significantly lyse rabbit erythrocytes, but human erythrocytes and sheep erythrocytes were insensitive to Hla killing effect. ELISA results showed that polyclonal antibodies with higher titer were obtained. The antibody can effectively inhibit Hla rabbit erythrocyte lysis. Conclusions The Hla protein of SAU has different ability to dissolve erythrocytes of different species, which may provide some clues for exploring the mechanism of Hla cell lysis. At the same time, the anti-Hla can inhibit the hemolysis of Hla, The pathogenesis has laid the foundation.