论文部分内容阅读
目的 :研究金黄色葡萄球菌 (金葡菌 )毒素的调控机制 ,构建pET_trap表达载体 ,在大肠杆菌中表达金葡菌毒素调控的重要分子TRAP蛋白。方法 :从金葡菌中提取基因组DNA ,用特异引物钓取trap基因。通过亚克隆的方法构建了pET_trap表达载体 ,在大肠杆菌中诱导表达 ,经亲和柱层析分离纯化。制备纯化蛋白的多抗血清 ,利用Western印迹检测多抗与天然蛋白的反应。结果 :目的基因在大肠杆菌中获得高表达 ,超声破碎后 ,表达产物主要存在于上清中 ,制备的多抗血清可以与天然蛋白发生特异性反应。结论 :通过原核表达获得了天然的TRAP蛋白 ,为研究金葡菌毒素调控机制及防治金葡菌感染奠定了基础
Objective: To study the regulation mechanism of Staphylococcus aureus (Staphylococcus aureus) toxin and to construct pET_trap expression vector to express Staphylococcus aureus important TRAP protein in Escherichia coli. Methods: Genomic DNA was extracted from S. aureus and the trap gene was captured by specific primers. The pET_trap expression vector was constructed by subcloning and expressed in E.coli. The recombinant plasmid was purified by affinity chromatography. Multi-antisera of purified protein were prepared and the reaction of polyclonal antibody with native protein was detected by Western blot. Results: The target gene was highly expressed in Escherichia coli. After sonicated, the expressed product mainly existed in the supernatant. The prepared polyclonal antiserum reacted specifically with the native protein. Conclusion: The natural TRAP protein was obtained by prokaryotic expression, which laid the foundation for the study of the regulation mechanism of Staphylococcus aureus and the prevention of S. aureus infection