Prokaryotic expression,purification and identification of low-toxic staphylococcal enterotoxin A with D227A mutation
10.3969/j.issn.1000-484X.2018.06.016
- VernacularTitle: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
- Author:
Xue-Ting LIU
1
;
Li-Ping ZENG
;
Yang XIE
;
Jian-Guo ZHANG
;
Ze-Hong ZOU
;
Ai-Lin TAO
Author Information
1. 广州医科大学附属第二医院
- Keywords:
SEA;
D227A mutation;
Prokaryotic expression;
Protein purification
- From:
Chinese Journal of Immunology
2018;34(6):882-886,891
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To obtain recombinant D227A mutation Staphylococcal enterotoxin A protein(rSEAD227A) with low toxicity but still retain its immunological activity and high purity. Methods: The SEA gene containing D227A mutation was cloned by PCR. By constructing pET44a-SEAD227Avector 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 D227A mutation of SEA was cloned and the expression system of Rosetta-rSEAD227Awas constructed. The purified rSEAD227Aprotein was obtained by refolding with gradient dialysis and affinity purification. LC-MS/MS analysis confirmed that the tryptic digested rSEAD227A peptide sequences matched the sequences of SEA in the database. Conclusion: The rSEAD227A protein in high purity was obtained,which provided the ex-perimental basis for further basic research and clinical application of SEA.
