Study On Fusion Expressions And Immunogenicity Of Different β、ε Toxin Fragement From Clostridium Perfringens

Clostridium perfringens is a Gram positive, non-motile, spore-forming bacterium, commonly found in soil, water, air, in the intestinal tract of animals and in a variety of raw and processed foods, which can induce zoonoses. Immunization is crucial for the control of the disease. Traditional vaccines againgst these diseases are mainly combination vaccines made of whole bacteria and toxoid. These vaccines has shortages like larger injection dose and obvious vaccination reactions which require further improvement of vaccine quality. In this study, the fusion genes of differentβtoxin and εtoxin was cloned and expressed respectively, then immunogenicity of the fusion proteins was analyzed to select protective antigens of each toxin, aimed at constructing a fusion proteain.According toβandεtoxin sequences published in GeneBank, The secondary structure of the toxin proteins was analyzed and predicted by DNAstar software. Different truncated genes of the βand εtoxin mature peptide were cloned into vector pET-32a(+) and expressed. Three fusion proteins obtained were named as ε1~432-β1~610、β419~927-ε430~889、β-ε respectively. The reactogenicity of fusion proteins were tested by Western blot. and their immunogenicity were tested for by direct toxin challenge in rabbits and toxin-antitoxin neutralization in mice. Results revealed that the protection induced by beta toxin protein depends on the integrity of whole toxin whereas truncated beta toxin alone can not. Main protective antigen of ε toxin is in the N terminal area 1~432 amino acid.According to above results, ε1~432-β fusion gene was constructed and expressed in E.coli. immunogenicity of the fusion protein ε1~432-β was tested by direct toxin challenge and toxin-antitoxin neutralization. Results showed that the fusion protein can provide 100% protection immunized animals from challenging of Clostridium perfringens type C toxin and induce higher titer of antitoxin, but can not protect the immunized rabbits from challenge of Clostridium perfringens type D toxin.We presumed that when construct a fusion protein of beta and epsilon toxin of Clostridium perfringens, a synergy or inhibitory effect may occur because of the toxin’s epitope structure. This phenomenon would only been observed when the length of the toxin is adaptive and further study is needed to prove this conclusion.