Construction,prokaryotic expression and identification of mutant SEA(D227A)
- VernacularTitle:点突变SEA(D227A)的基因构建、原核表达与鉴定
- Author:
Jing YE
;
Yanfang SUI
;
Guangsheng CHEN
;
Zengshan LI
;
Xiumin ZHANG
;
Yunxin CAO
- Publication Type:Journal Article
- Keywords:
SEA;
Superantigen;
Prokaryotic expression;
Mutant
- From:
Chinese Journal of Immunology
2000;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the prokaryotic expression vector of SEA mutant gene SEA(D227A).The gene was expressed in E.Coli, and the induced protein was purified and identified.Methods:The SEA gene was cloned by PCR from Staphylococcus aureus strain FRI 100.D227A was introduced by changing the Asp codon GAT into GCT(Ala)in the primer.The expression plasmid pRSET SEA(D227A)was constructed and transformed into E.Coli BL21(DE3)pLysS.The induced protein was identified by Western blot.Results:The nucleotide sequence of SEA(D227A)was found to be identical to the designed sequence. E.Coli BL21(DE3)pLysS contained pRSET SEA(D227A)can express a 32 kD protein which can specially bind with the anti SEA mAb.The induced protein was purified with Ni 2+ system.Conclusion:The SEA(D227A)gene was constructed and expressed successfully.The study gave a clue to the research of low toxic superantigen. [
