In order to amplify alpha toxin gene of Clostridium septicum HeB01 strain, one pair of primers was designed according to the GenBank sequence, and a 1323bp alpha toxin gene fragment was obstained by PCR. Sequence analysis indicated that the homology of the nucleotid sequence of HeB01 strain to those other reference strains was more than 99.5% . The expression plasmid pQE30-alpha was constructed by inserting alpha toxin gene into the prokaryotic expression vector pQE30. The plasmid expressed when the recombinant strain M15(pQE30-alpha) was induced by IPTG. The specific 48 kD protein was detected SDS-PAGE and the immunogenicity of the expressed alpha toxin was confirmed by Western blot and ELISA. The expressed alpha toxin was transformed into alpha toxoid vaccine by adding 0.3% formaldehyde into alpha toxin. The protective immune response was proved after the mice was immunized with alpha toxoid vaccine. The results showed that the recombinanted strain M15 (pQE30-alpha) could be as a candidate of alpha toxoid vaccine to provide protective immune response against clostridium septicum infection.
Animals ; Antibodies, Bacterial ; blood ; immunology ; Bacterial Toxins ; genetics ; immunology ; metabolism ; Blotting, Western ; Cloning, Molecular ; Clostridium Infections ; immunology ; microbiology ; prevention & control ; Clostridium septicum ; genetics ; immunology ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Gene Expression Regulation, Bacterial ; Mice ; Polymerase Chain Reaction ; Recombinant Proteins ; administration & dosage ; immunology ; metabolism ; Toxoids ; immunology ; Vaccination