Prokaryotic expression,purification and identification of low-toxic staphylococcal enterotoxin A with D227A mutation / 中国免疫学杂志
Chinese Journal of Immunology
; (12): 882-886,891, 2018.
Article
em Zh
| WPRIM
| ID: wpr-702836
Biblioteca responsável:
WPRO
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.
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Índice:
WPRIM
Tipo de estudo:
Diagnostic_studies
Idioma:
Zh
Revista:
Chinese Journal of Immunology
Ano de publicação:
2018
Tipo de documento:
Article