Abstract: Epsilon-toxin (ε-toxin) gene was amplified from chromosomal DNA of Clostridium perfringens type D (C60-2) by polymerase chain reaction (PCR),and a 906 bp epsilon toxin gene fragment was obtained. Sequence analysis indicated that the homology of the nucleotide sequence of the strain to those other reference strains was more than 99%. The expression plasmid pET32a-ETX was constructed by inserting the epsilon toxin gene into the prokaryotic expression vector pET32a. The plasmid pET32a-ETX was transformated into E.coli BL21(DE3)plys and the recombinant strain BL21(pET32a-ETX) was obtained. Then,the transformants were induced to express with IPTG. The specific 54 ku protein was detected by SDS-PAGE and the immunogenicity of the expressed epsilon toxin was confirmed by Western blotting and ELISA. The obtained recombinant protein was transformed into epsilon toxoid vaccine by adding 0.4% formaldehyde into epsilon toxin. The protective immune response was proved after the mice was immunized with epsilon toxoid vaccine. The results showed that the recombinanted strain BL21(pET32a-ETX) could be as a candidate of epsilon toxoid vaccine to provide protective immune response against C. perfringens type D infection.

Key words: Clostridium perfringens type D; epsilon-toxin; toxoid; immunoprotection