【摘要】 以提取的奶牛乳腺炎源性金黄色葡萄球菌山东分离株(zfb)全基因组DNA为模板,PCR扩增β-溶血素基因(hlb),并与克隆载体pMD18-T相连接。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,与已报道的金黄色葡萄球菌β-溶血素蛋白的氨基酸同源性为99.4%。构建原核表达载体pET32a+/hlb,SDS-PAGE分析蛋白表达水平,IPTG诱导后表达的融合蛋白的相对分子量质为57000,表达量占菌体总蛋白的23.9%;表明原核表达质粒构建成功,并实现了有效表达。更多还原

【Abstract】 Beta-hemolysin gene was cloned from genome DNA of Staphylococcus aureus strains Shandong of China by PCR, and the PCR product was inserted into vector pMD18-T. Sequence analysis showed that the Staphylococcus aureus β-hemolysin gene (hlb) was composed of 993 nucleotides encoding a mature polypeptide 330 amino acids. Compared to other reported hlb,it shared the identity of 99.4%. The recombinant plasmid pET32a+/hlb was constructed, and the expression of hlb in Escherichia coli was induced by addition of IPTG. SDS-PAGE results showed that recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 57 000 and the recombinant proteins accounted for 23.9% of the whole proteins. The results indicated that the expression plasmid was successfully constructed and hlb protein was expressed in Escherichia coli, and this study provides conditions for the research of β-hemolysin pathogenic and immune mechanism and vaccine design.更多还原