中国生物工程杂志

Abject To construct an α-hemolysin (α-HL)gene deficient S . aureus strains with the ability to produce staphylococcal enterotoxin B(SEB), and then to obtain the deletion mutation with the same gene background except α-HL gene with wild-type strain. Plasmid pMHL-α was constructed for homologous recombination of the α-HL system of S . aureus by inserting the neomycin resistance gene expressing unit and two α-HL -flanking regions into a shuttle plasmid pMAD. The recombination vector pMHL-α was constructed correctly by restriction analysis and PCR. Because S . aureus SM-01 has no ability to accept foreign DNA, the plasmid pMHL-α was firstly introduced by electroporation into restriction-deficient laboratory strain RN4220 and then transformed to the S . aureus SM-01 by protoplast transformation. Because the recombination vector is temperature sensitive for DNA replication, the wild-type strain SM-01 with recombination vector was incubated at the nonpermissive temperature while maintaining selective pressure to select the mutation of S. aureus α-HL delection strain. The deletion mutation of α-HL was proved by the antibiotic resistance, PCR analysis and the hemolysis assay. S. aureus α-HL deletion mutation was successfully constructed by homologous recombination with the main coding sequences of S . aureus α-HL were replaced by neomycin resistance gene. It could be of great value for providing some theories and approach to study genetic characters of S .aureus and construct genetically engineered bacteria for producing superantigen.