摘要
目的利用大肠杆菌重组表达金黄色葡萄球菌外分泌毒素β-溶血素(β-hemolysin,Hlb)及其突变体HlbH-149-N,检测其生物学活性,并制备其功能性抗体,探讨Hlb蛋白在金葡菌感染中的生物学意义。方法以金葡菌NCTC-8325基因组为模板,利用PCR技术扩增目的基因hlb,进一步构建重组表达载体p ET28a-hlb,并转至大肠杆菌BL21(DE3),利用点突变技术构建重组表达载体p ET28a-hlbH-149-N,经IPTG诱导表达目的蛋白,通过Ni2+柱亲和纯化获得Hlb蛋白及突变体HlbH-149-N蛋白;通过溶血实验检测Hlb及突变体HlbH-149-N蛋白的生物学活性,并制备Hlb特异性抗体,检测抗体的中和活性。结果获得了纯度较高的重组Hlb蛋白和突变体HlbH-149-N蛋白,溶血活性实验结果表明,重组Hlb蛋白能够裂解绵羊红细胞,而突变体HlbH-149-N蛋白不能裂解绵羊红细胞;不同物种红细胞对Hlb敏感性不同;制备了抗Hlb蛋白的多克隆抗体(anti-Hlb),ELISA和Western印迹实验结果表明,anti-Hlb能特异结合Hlb蛋白和突变体HlbH-149-N蛋白,同时发现,anti-Hlb可有效抑制Hlb对绵羊红细胞的裂解作用。结论获得了具有良好溶血活性的重组Hlb蛋白以及无溶血活性的突变体HlbH-149-N蛋白,并制备了具有中和活性的特异性抗体,为深入研究Hlb在金葡菌致病过程中的作用奠定了基础。
Objective To express the beta hemolysin （Hlb）, an important toxin secreted by Staphylococcus aureus （S. aureus） and the mutant protein HlbH-149-N, to detect the hemolytic activity of Hlb and HlbH_149_N on sheep erythrocytes, and to prepare the specific antibodies against Hlb which can inhibit the hemolytic activity of Hlb. Methods Hlb gene was amplified by PCR using S. aureus NCTC-8325 genome DNA as template. The expression vector pET-28a-hlb was constructed and transformed into E. coli BL21 （DE3）. The expression vector pET28a-hlbH-149-N was constructed through point mutation. The recombinant Hlb and HlbH-149-N protein were expressed and purified by Ni^2＋ affinity chromatography. The hemolytic activity of Hlb and HlbH-149-N was measured by sheep erythrocyte lysis assay. Results Recombinant Hlb protein and the mutant were obtained. Further investigations showed that Hlb could significantly induce the lysis of SRBC while HlbH-149-N could not. The specific polyclonal antibodies against Hlb （anti-Hlb） were prepared. It was found that anti-Hlb recognized Hlb and HlbH-149-N,Moreover, it was found that anti-Hlb blocked the hemolytic activity of Hlb. Conclusion The recombinant Hlb protein with high hemolytic activity and HlbH-149-N without hemolytic activity are obtained while its neutralized antibody is pepared. Hlb from S. aureus has different hemolytic effects on erythrocytes from various species. Our findings will facilitate the investigation on the role of Hlb in the pathogenesis of S. aureus.
出处
《军事医学》
CAS
CSCD
北大核心
2016年第2期127-132,共6页
Military Medical Sciences
基金
国家自然科学基金面上资助项目(31370170
31170074)
关键词
葡萄球菌
金黄色
β-溶血素
溶血活性
抗体制备
Staphylococcus aureus
beta hemolysin
hemolysin activity
antibody preparation
