Study on expression and functions of staphylococcal enterotoxin B mutants
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 S 6B . 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, T 150 A). As being expected, at the residue 23 of SEB M, serine substituted for asparagine (AAT→AGT, N 23 S) 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 T 150 A and N 23 S 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.