低毒性金葡萄球菌肠毒素A D227A突变体的原核表达、纯化及鉴定  

Prokaryotic expression,purification and identification of low-toxic staphylococcal enterotoxin A with D227A mutation

作　　者：刘雪婷[1] 曾丽萍[1] 谢洋 张建国[1] 邹泽红[1] 陶爱林[1] LIU Xue-Ting;ZENG Li-Ping;XIE Yang;ZHANG Jian-Guo;ZOU Ze-Hong;TAO Ai-Lin(The Second Affiliated Hospital of Guangzhou Medical University, Guangdong Province Key Laboratory of Allergy ＆ Clinical Immunology, Allergy Research Branch of the State Key Laboratory of Respiratory Disease, Guangzhou 510260, China)

机构地区：[1]广州医科大学附属第二医院广东省过敏反应与免疫重点实验室呼吸疾病国家重点实验室,广州510260

出　　处：《中国免疫学杂志》2018年第6期882-886,891,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金(81301948;30640033);广州市过敏反应临床医学研究与转化中心建设项目(穗科创字[2016]171号)的资助

摘　　要：目的:获得低毒性但仍保留其免疫活性、高纯度的重组D227A突变型金葡菌肠毒素A蛋白(rSEA_(D227A))。方法:利用PCR技术克隆含D227A突变的SEA基因并原核表达,用盐酸胍溶解包涵体并进行梯度透析复性;使用StrepⅡ亲和层析来纯化蛋白;免疫印迹和LC-MS/MS进行鉴定。结果:成功克隆SEA的D227A突变体,获得了高纯度的rSEA_(D227A)蛋白。LCMS/MS分析证实,胰酶消化后的rSEA_(D227A)肽段序列与数据库中SEA的序列匹配。结论:获得了高纯度rSEA_(D227A)蛋白,为SEA的进一步基础研究和临床应用提供了实验基础。Objective：To obtain recombinant D227 A mutation Staphylococcal enterotoxin A protein（rSEA_（D227A）） with low toxicity but still retain its immunological activity and high purity.Methods：The SEA gene containing D227 A mutation was cloned by PCR.By constructing p ET44 a-SEAD227 Avector and transfecting the expression strain Rosetta,inclusion bodies were solubilized with guanidium hydrochloride and refolded by gradient dialysis;proteins were purified using StrepⅡ affinity chromatography,and identified by Western blot and high performance liquid chromatography-mass spectrometry（LC-MS/MS）.Results：The D227 A mutation of SEA was cloned and the expression system of Rosetta-rSEA_（D227A） was constructed.The purified rSEA_（D227A） protein was obtained by refolding with gradient dialysis and affinity purification.LC-MS/MS analysis confirmed that the tryptic digested rSEA_（D227A） peptide sequences matched the sequences of SEA in the database.Conclusion：The rSEA_（D227A） protein in high purity was obtained,which provided the experimental basis for further basic research and clinical application of SEA.

关 键 词：SEA D227A突变 原核表达 蛋白纯化 

分 类 号：R392.8[医药卫生—免疫学]