关键词: 金黄色葡萄球菌, β-溶血素, 克隆, 表达, 溶血活性, CAMP反应

Abstract:

【Objective】 The gene coding the beta-hemolysin was cloned from Staphylococcus aureus Shandong strain Zfb, expressed in Escherichia coli, and the hemolytic activity of the recombinant protein was analyzed. 【Method】 hlb gene was cloned from genomic DNA of Staphylococcus aureus by PCR, and the PCR product was inserted into vector pET32a+ to construct plasmid pET32a+/hlb, the plasmid was then expressed in Escherichia coli BL21 (DE3) cells that induced by IPTG.. The hemolytic activity was evaluated by 96 well microtiter V-plates using purified the recombination protein against sheep and bovine erythrocytes and CAMP reaction with Streptococcus agalactiae. 【Result】 Sequence analysis showed that the Staphylococcus aureus β-hemolysin gene (hlb) was composed of 993 nucleotides encoding a mature polypeptide 330 amino acids; SDS-PAGE results showed that the recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 57 kD and the recombinant proteins accounted for 23.9% of the whole proteins. The purified protein hlb has 278 HU?mg-1 to sheep erythrocytes and 9×103 HU?mg-1 to bovine erythrocytes. The purified protein hlb can make obviously CAMP reaction with Streptococcus agalactiae. 【Conclusion】 The results showed that hlb protein was well expressed in Escherichia coli, and the hemolytic activity to bovine erythrocytes is bigger than sheep erythrocytes, suggesting that β- hemolysin pathogenic，immune mechanism and mastitis vaccine design might be further researched. Also, a new method of specific diagnosis and discriminating Streptococcus agalactiae from mastitis cases pathogen using the recombinant proteins by CAMP reaction is established.

Key words: Staphylococcus aureus, β-hemolysin gene, cloning, expression, hemolytic activity, CAMP reaction