牛乳中金黄色葡萄球菌山东分离株(zfb)β-溶血素(hlb)的克隆、表达及溶血活性分析

Expression of β-hemolysin Gene of Staphylococcus aureus Strains Shandong(zfb) and Its Hemolytic Activity

【目的】实现牛乳中金黄色葡萄球菌β-溶血素在大肠杆菌中的高效表达,并分析重组β-溶血素的溶血活性。【方法】PCR扩增牛乳中金黄色葡萄球菌山东分离株β-溶血素基因,构建原核表达载体pET32a+/hlb,并在大肠杆菌BL21(DE3)中诱导表达,用96孔血凝板测定重组蛋白的溶血效价,以血琼脂平板法检测重组蛋白的CAMP反应。【结果】测序结果表明,扩增片段含有993bp的ORF,可编码含330个氨基酸的成熟蛋白;SDS-PAGE分析重组蛋白表达水平,IPTG诱导后表达的融合蛋白分子量为57kD,表达量占菌体总蛋白的23.9%;溶血效价分析,金葡菌β-溶血素对绵羊红细胞的溶血效价为278HU·mg-1,对奶牛红细胞的溶血效价为9×103HU·mg-1;重组蛋白在血琼脂平板上和无乳链球菌能发生明显的CAMP反应。【结论】成功表达了牛乳中金黄色葡萄球菌β-溶血素,该毒素对奶牛红细胞的溶血活性明显大于绵羊红细胞,显示出牛源金黄色葡萄球菌β-溶血素的特性明显不同于人源金黄色葡萄球菌β-溶血素,为进一步研究其致病与免疫机理、疫苗设计提供了理论基础。此外,重组蛋白还可用来特异性诊断、鉴别无乳链球菌,为兽医临床诊断提供新的方法。

[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 9×10^3 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.