Prokaryotic expression of exfoliative toxin A from Staphylococcus aureus

Abstract:

Objective：To obtain the recombine exfoliative toxin A（rETA） by the technique of gene engineering and to provide materi－als to study the pathogenic mechanism of the exfoliative toxin（ET）. Methods：The primers were designed according to the gene se－quence of ETA gene announced by GeneBank. The ETA gene was amplified from the genome of S.aureus and was inserted into the prokaryotic expression vector pQE30. The recombinant plasmid pQE30-ETA was built and was translated into E.coli M15. After the expression conditions being optimized，the recombinant plasmid pQE30-ETA was expressed. The expression product was analyzed by SDS-PAGE and confirmed by Western blot. After being purified with Ni2+-NTA resin，the purified protein was injected into the sub－cutaeous of BALB/C mice and pathological modifications of BALB/C mice were observed to identify the biologic activity of the rETA. Results：The gene sequence analysis of the recombinant plasmid pQE30-ETA showed that the inserted gene fragment was 927 bp and was corresponded with the ETA gene sequence in GenBank database. The analysis of SDS-PAGE showed that the relative molecular mass of expression was 27 kD and the recombinant solubility protein reached the highest expression（20%） when IPTG being at a fi－nal concentration of 0.1 mmol/L and the induction time of 4 h. Western blot showed that the recombinant protein was at the same lo－cation with the ETA protein produced by S.aureus. The purity of the recombinant protein was over 90% after being purified with Ni2+-NTA resin. The splitting and blister were appeared in the epidermis of mouse injected with recombinant protein. Conclusion：The construction of the recombinant plasmid pQE30-ETA and the expression of rETA with biologic activity are successful.