金黄色葡萄球菌α-溶血素的表达、纯化及中和性抗体的制备

Expression,purification and antibody preparation of alpha-hemolysin from Staphylococcus aureus

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 clone and express alpha hemolysin （Hla）, one important virulence factor secreted by Staphylococcus aureus in Escherichia coli to tdetect the hemolytic activity of Hla on erythrocytes from diffrerent species, and to prepare specific antibodies against Hla that can inhibit the hemolytic activity of Hla. Methods Hla gene was amplified by PCR using S. aureus NCTC-8325 genome DNA as a template. The expression vector pET-28a-Hla was constructed and transformed into E. coli BL21 （DE3）. The recombinant Hla protein was expressed by IPTG induction, and then purified by Ni^2 ＋affinity chromatography. The hemolytic activity of Hla was measured by erythrocyte lysis assays. Results Soluble recombinant Hla protein was expressed and purified. Further investigations showed that Hla could significantly induce the lysis of rabbit erythrocytes. However, erythrocytes from humans or sheep were more resistant to the lysis mediated by Hla. The specific polyclonal antibodies against Hla （anti-Hla） were prepared and detected by ELISA. Moreover, it was found that anti-Hla could inhibit the hemolytic activity of Hla. Conclusion We found that Hla from S. aureus has different hemolytic effects on erythrocytes from various species. The prepared Hla-antibodies can specifically block the hemolytic activity of Hla.