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

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.