金黄色葡萄球菌β-溶血素的表达、纯化及中和性抗体的制备

发布时间：2017-12-27 12:33

本文关键词：金黄色葡萄球菌β-溶血素的表达、纯化及中和性抗体的制备　出处：《军事医学》2016年02期 　论文类型：期刊论文

【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。
[Abstract]:Objective to detect the biological activity of Staphylococcus aureus exotoxin -hemolysin (Hlb) and its mutant HlbH-149-N by recombinant E.coli, and to prepare its functional antibody, and to explore the biological significance of Hlb protein in Staphylococcus aureus infection. Methods Staphylococcus aureus genomic NCTC-8325 as template, HLB amplification of the target gene by PCR technology to construct the recombinant expression vector p ET28a-hlb, and transferred to E.coli BL21 (DE3), using ET28a-hlbH-149-N technology to construct the recombinant expression vector p mutation induced by IPTG protein expression, Hlb protein and mutant HlbH-149-N protein purified by Ni2+ affinity; biological activity by hemolysis test to detect the expression of Hlb and mutant HlbH-149-N protein, and preparation of Hlb antibody, neutralizing antibody. Results the recombinant Hlb protein and mutant HlbH-149-N protein with high purity, hemolytic activity test results showed that the recombinant Hlb protein could lyse sheep red blood cells, while HlbH-149-N mutant protein can cleavage of sheep red blood cells; erythrocytes of different species in different sensitivity to Hlb; polyclonal antibody against Hlb protein was prepared (anti-Hlb), and ELISA Western blot results showed that anti-Hlb can bind Hlb protein and mutant HlbH-149-N protein, also found that anti-Hlb can effectively inhibit the lysis of sheep erythrocytes Hlb. Conclusion a recombinant Hlb protein with good hemolytic activity and a HlbH-149-N protein without hemolytic activity were obtained, and a specific antibody with neutralization activity was prepared, which laid a foundation for further research on the role of Hlb in the pathogenesis of Staphylococcus aureus.
【作者单位】：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】：R392
【正文快照】： 金黄色葡萄球菌(Staphylococcus aureus,金葡菌)是一种自然界中广泛存在的革兰阳性菌,可引起人和动物的局部化脓性感染、肺炎、伪膜性肠炎、心包炎以及败血症、脓毒败血症等,同时也是造成人类食物中毒的常见致病菌之一[1]。金葡菌主要通过产生多种致病因子而致病,这些致病因子

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