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

发布时间：2019-02-12 18:02

【摘要】：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。
[Abstract]:Objective to express 伪 -hemolysin (alpha hemolysin,Hla) of staphylococcus aureus by recombinant expression of 伪 -hemolysin (alpha hemolysin,Hla), an important exocrine of Staphylococcus aureus, and to detect the lytic effect of Hla protein on erythrocytes of different species. The specific antibody was prepared and the neutralization activity of the antibody was detected. Methods using the genome of SAU NCTC-8325 strain as template, the hla gene was amplified by PCR. The recombinant expression vector p ET-28a-Hla, was constructed and transformed into Escherichia coli BL21 (DE3). Hla protein was obtained by IPTG induced expression and purified by Ni2 column affinity. The hemolytic activity of Hla protein was determined by erythrocyte lysis test. Results Recombinant Hla protein was expressed in soluble form. Cell lysis assay showed that the purified Hla protein could significantly split rabbit erythrocytes, but human erythrocytes and sheep erythrocytes were not sensitive to the killing effect of Hla. A polyclonal antibody with high titer was obtained. The antibody can effectively inhibit the effect of Hla on rabbit erythrocyte lysis. Conclusion the Hla protein of SAU has different ability to dissolve erythrocytes in different species, which may be helpful for the study of the mechanism of Hla lysis cells. At the same time, the specific antibody (anti-Hla) can inhibit hemolysis of Hla, which lays a foundation for further study of the pathogenesis of Hla.
【作者单位】：广西医科大学基础医学院;军事医学科学院基础医学研究所;
【基金】：国家自然科学基金面上资助项目(31370170,31170074)
【分类号】：R378.11

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