金黄色葡萄球菌肠毒素A（SEA）多克隆抗体的制备及鉴定

目的：制备金黄色葡萄球菌肠毒素A（SEA）多克隆抗体并用金黄色葡萄球菌野生株对其进行验证,为进一步研究SEA在金黄色葡萄球菌致病机制中的作用提供基础。方法：PCR方法扩增肠毒素A基因（sea）,构建原核表达载体p ET 30 a/sea,经PCR方法和测序鉴定后,阳性表达载体转化大肠杆菌表达宿主菌DH 5α,表达产物用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹法（Western Bloting,WB）分析鉴定。蛋白经纯化后作为免疫原免疫新西兰大白兔,得到抗血清并纯化,采用sea基因阳性的金黄色葡萄球菌野生株S8和S 23株的菌体蛋白进行鉴定。结果：pET 30 a/sea重组表达载体构建成功,目的蛋白在构建的原核表达系统中可实现高表达,纯化后得到高纯度的SEA蛋白,经临床分离金黄色葡萄球菌S 8和S 23株菌体蛋白验证,证实得到理想的兔抗SEA多克隆抗体。结论：通过基因重组技术,金黄色葡萄球菌SEA蛋白在构建的原核表达系统中得以成功表达,并获得了高纯度的目的蛋白及其多克隆抗体,为进一步研究金黄色葡萄球菌肠毒素A蛋白的生物学活性及抗体的保护作用奠定了基础。

PREPARATION AND IDENTIFICATION OF ANTI-STAPHYLOCOCCUS AUREUS ENTEROTOXIN A(SEA) POLYCLONAL ANTIBODY

Objective： To prepare staphylococcus aureus enterotoxin A（ SEA） polyclonal antibody and verify it using staphylococcus aureus clinical isolates,and further provide basis for exploring the effects of SEA in the pathogenesis of staphylococcus aureus. Methods： PCR method was used to amplify enterotoxin A gene（ sea）,and then the prokaryotic expression vector p ET 30 a / sea was constructed.The constructed vector were verified by PCR method and gene sequencing method,respectively. The positive vectors were transformed into Escherichia coli expression strain,DH 5α strain. The expression products were identified by Twelve sodium dodecyl sulfate- polyacrylamide gel electrophoresis（ SDS-PAGE） and western blotting technique（ WB）. The purified protein was used as immunogen to immune New Zealand white rabbits and the anti- sera was obtained,purified and identified using mycoprotein abstracted from clinical staphylococcus aureus isolates S8 and S23. Results： p ET 30 a / sea recombination expression vector was successfully constructed and the target protein was highly expressed in prokaryotic expression system,and the highly purified SEA protein was verified by mycoprotein abstracted from clinical staphylococcus aureus isolates S8 and S23,which confirmed that the ideal anti-SEA antibody was obtained. Conclusion： staphylococcus aureus enterotoxin A gene was successfully expressed in the constructed prokaryotic expression system and the highly purified target protein and its polyclonal antibody,which provide a basis for further investigating the biological activities of staphylococcus aureus enterotoxin A protein and the protective effects of its polycolonal antibody.