Study on expression and functions of staphylococcal enterotoxin B mutants
- VernacularTitle:葡萄球菌肠毒素B突变体基因表达和功能的初步研究
- Author:
Hong YU
;
Lisheng QIAN
- Publication Type:Journal Article
- From:
Chinese Journal of Microbiology and Immunology
2001;21(2):200-203
- CountryChina
- Language:Chinese
-
Abstract:
Objective To obtain staphylococcal enterotoxin B (SEB) mutant with normal antigenicity but low toxicity. Methods Using PCR technique, normal SEB (SEB-N) gene which was amplified from S. aureus S6B. SEB mutant gene (SEB-M) was prepared from the same strain, but one nucleotide in SEB gene was changed from asparagine (N23) to serine (S23). SEB-N and SEB-M were cloned into procaryotic expression vector pTrc99A then and transferred into E. coli JM109. SEB-N and SEB-M which were cloned into plasmid were sequenced directly by dideoxynucleotide method. The crude expressed proteins were identified by double agar immunodiffusion. The level of IL-2 in supernatants of mouse splenocytes stimulated by crude expressed proteins was determined by ELISA. Results SEB-N and SEB-M were obtained through PCR. The sequence of SEB-N was changed with non site-directed mutagenesis, threonine at the residue 150 of SEB-N was replaced with alanine (ACT→GCT, T150A). As being expected, at the residue 23 of SEB-M, serine substituted for asparagine (AAT→AGT, N23S) with site-directed mutagenesis. Double agar immunodiffusion showed obvious precipitin line with anti-SEB by both crude SEB-N and SEB-M mutant proteins could produce, but not by non-recombinant strain. ELISA demonstrated that the level of IL-2 in supernatant of mouse splenocytes stimulated by natural SEB protein (containing equal amount of JM109P crude protein) was 40 times as much as that stimulated by SEB-M and 12.5 times as much as that stimulated by SEB-N. Conclusions We obtained two recombinant strains which produced T150A and N23S mutant SEB protein. The mutant proteins showed binding ability to anti-SEB as the normal protein. However, their biological activity as superantigen decreased sharply. We consider that it is promising for further study of molecular adjuvant or superantigen vaccine.
