【摘要】 【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。更多还原

【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 vaccinedesign 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.更多还原