[关键词]
[摘要]
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pET 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S
8 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。
[Key word]
[Abstract]
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 pET 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( SDSPAGE)
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: pET 30a /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．
[中图分类号]
Ｒ37
[基金项目]
?收稿日期:2014－09－01; 修回日期:2014－10－02
基金项目:国家自然科学基金( 81260244)
作者简介:王俊瑞( 1981－) ，男，内蒙古医科大学附属医院检验科主治医师。
通讯作者:韩艳秋，主任医师，硕士研究生导师，E－mail: qYH1016@ sina． com 内蒙古医科大学附属医院， 010050
