Molecular Cloning And Prokaryotic Expression Of Enterotoxin B Gene Of Staphylococcal Aureus Clinical Isolates In Escherichia Coli DH5α

Objective To construct prokaryotic expression vector of staphyloc-occal aureus enterotoxin B gene, express it in Escherichia coli DH5α, and study the biological activities of the fusion proteins.Methods Total DNA was extracted from staphylococcus aureus and the DNA fragment encoding mature peptide precursor of staphylococcal enterotoxin B was amplified by PCR, with the aid of DNAstar Softeware for primers design. The fragment was cloned into the multiple cloning site (MCS) of glutathione S-transferase(GST) of plasmid pGEX-4T-2, forming the recombinant prokaryotic expression vector (pGEX-4T-2/SEB). The recombinant plasmid (named pGEX-4T-2/SEB) was identified by restrictive endonuclease assay and its sequence was determined by dideoxy-chain termination. The recombinant plasmid pGEX-4T-2/SEB was transformed into E.coli DH5αby TSS and induced by Isopropyl-β-D-thioglactoside (IPIG) to express GST/SEB fusion protein. Then the expressed proteins in E.coli DH5αwere analyzed with SDS-PAGE electrophoresis. Western blotting was performed to identify the fusion protein .Results The recombinant plasmid (pGEX-4T-2/SEB) was obtained and its opening read frame(ORF) was verified by sequence analysis. The results of SDS-PAGE showed that there was a noval protein band, which was 58×10~3 Daltons (relative molecular weight, Mr). Western blotting assay proved that the fusion protein could be specifically recognized by anti-SEB antibody.Conclusions These results showed that mature peptide precursor gene of staphylococcal enterotoxin B was efficiently expressed in E.coli DH5αand the previous work has not only roll out a basis for the studies of its biological activities, but also laid a necessary foundation for future research on possible clinic applications in the future.