Expression, purification and antibody preparation of beta-hemolysin from Staphylo-coccus aureus
10.7644/j.issn.1674-9960.2016.02.013
- VernacularTitle: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
- Author:
Chenghua LIU
;
Di LI
;
Yaping GAO
;
Peng JIN
;
Yu LIU
;
Guang YANG
- Publication Type:Journal Article
- Keywords:
Staphylococcus aureus;
beta hemolysin;
hemolysin activity;
antibody preparation
- From:
Military Medical Sciences
2016;40(2):127-132
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express the beta hemolysin ( Hlb), an important toxin secreted by Staphylococcus aureus ( S.aureus) and the mutant protein Hlb H-149-N , to detect the hemolytic activity of Hlb and Hlb H-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-Nwas constructed through point mutation.The recombinant Hlb and Hlb H-149-N protein were expressed and purified by Ni 2+affinity chromatography .The hemolytic activity of Hlb and Hlb H-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 Hlb H-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 Hlb H-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.
