The Fermentation Technology Study On Clostridium Perfringens Exotoxin And The Study Of The Toxoid Oil Emulsion Vaccine

The"sudden death"which caused by Clostridium perfringens have made a great damage to the animal husbandry development,so the criticality of immunoprophylaxis is to produce toxoid vaccine according the pathomechan- ism of the toxin.By using utomatic mechanical stirring fermenter,the toxin fermentation technology of ATCC type A Clostridium perfringens was studyed. The toxoid oil emulsion vaccine was produced and its immunopoten- cy was also investigated and made a comparation to the commercialization bacterium vaccine.It was conclude that pH7.0, at 43℃, and cultivated for 6h was the best condition for toxin fermentation and the toxoid vaccine was effective for animal immunity and better than the inactivated bacterium vaccine.This research consists of three parts.PartⅠ: The study of fermentation technology onα-toxin of type A Clostridium perfringens.By using automatic mechanical stirring fermenter, three factors, including temperature, pH value, and fermentation time were utilized in L9(34)orthogonal experiment in order to ascertain which zymotechnique was the most important factor in producingα-toxin and the growth and toxin production curves of Clostridium perfringens were drawn. The results indicated that pH value was significant for toxin producing (0.010.05). So, the best fermentation process of type A Clostridium perfringens in producingα-toxin was in pH7.0, at 43℃, and cultivated for 6h.PartⅡ: The study research of the Clostridium perfringens type A toxoid oil emulsion vaccine and the responses of the rabbits after immunizedTheα-toxin of type A Clostridium perfringens producing by using automatic mechanical stirring fermenter was inactivated by 0.4%formaldehyde and the Clostridium perfringens type A toxoid oil emulsion vaccine was successfully produced. The serum antibody growth and decline regulation and the immune protective efficiency of the rabbits was studied. It was also compared with inactivated bacterium vaccine. After series of detecting tests, the toxoid vaccine was accord with the manufacture criterion. The serum antibody of rabbits was reached the highest titer with 8.4log2 of antitoxin and 8.6log2 of anti-bacterium at the fourth week and it was respectively 6.5log2和5.4log2 at the 19th week after immunized by 2 mL, and the immune protective efficiency was 100% from 2 to 19 weeks. The serum antitoxin of rabbits immuned toxoid vaccine was significantly higher than the ones immuned inactivated bacterium vaccine(P<0.05), although there was no significant difference in anti-bacterium titer(P>0.05), and the same as the animal safety and immune protective efficiency. It was conclude that the toxoid vaccine was effective for animal immunity.PartⅢ:The improvement of the method about purifying theα-toxin of Clostridium perfringens type ADuiring the purification of the Clostridium perfringens type A toxin, the ammonium sulfate of 60% degree of saturation was used to salting out the toxin,then the Amicon Ultra-15 ultrafiltrate centrifuge tube was used to purify the initial extracted toxin after the procedure of dialysis and the initial extracted and purifyed toxin were treated by SDS-PAGE. It could be conclude that the highly purified toxin could be obtained through the protein level determination and the SDS-PAGE,and the content of the purified toxin was about one-fifth of the initial extracted toxin.