Molecular cloning of staphylococcal enterotoxin gene from wild Staphylococcus aureus strains

Objective To clone novel staphylococcal enterotoxin P(SEP)gene from Staphylococcus aureus(S.auresu)strains SD and 104 for the production of a novel staphylococcal enterotoxin. Methods Primers were designed according to the conserved sequence of SEP from published data.PCR products amplified from genomic DNA of S.aureus SD and 104 were inserted into the plasmid pET-28a.The resultant plasmids were transformed into E.coli BL21(DE3)and induced to express the gene with isopropyl-β-D-thiogalactoside(IPTG).Proteins purified through nickel-nitrilotriacetic acid(Ni⁺-NTA) affinity chromatography were used to analyze the superantigenic activity and anti-tumor effect in vitro. Results Two novel staphylococcal enterotoxin P genes were obtained according to the results of sequence alignment.E.coli harboring pET28aSEP_SD and pET28a-SEP₁₀₄ could high-efficiently express the soluble protein under 1 mmol/L IPTG induction.The proteins were purified through Ni⁺-NTA affinity chromatography.The purified protein was used to stimulate peripheral blood mononuclear cell(PBMC)proliferation.Compared with staphylococcal enterotoxin C2(SEC2),the proliferation rates of the proteins were higher than 30%.SEP could obviously inhibit the growth of tumor cells,even higher than SEC2. Conclusion New novel SEP genes were successfully cloned and the expressed proteins SEP_SD and SEP₁₀₄ of the genes exhibited anti-tumor effect stronger than SCE2.