| Clostridium septium as the Gram-positive anaerobic bacillus can cause diseases to animals andhumanbeings such as malignant edema, muscle necrosis, gas gangrene, necrotizing enteritis and so on,and Clostridium septium is also the pathogen of sheep braxy. The bacterium can secrete many kinds ofexotoxins such as、β、γ and, and its main pathogenic factor and immune protective antigen is-toxinwhich has hemolysis, necrosis and lethal activity.According to the gene sequences of Clostridium septicum-toxin in GenBank, PCR primers weredesigned, and-toxin gene (csa) and two truncated-toxins genes (csan219and csac439) wereamplified. These three gene sequences were inserted into pET-28a(+), and the expression vectors wereconstructed and transformed into E.coli BL21(DE3). The recombinant E.coli BL21(pET28a-csa),BL21(pET28a219) and BL21(pET28a439) were identified by PCR, double enzyme digestion andsequencing. The study results confirmed that these expression vectors were constructed successfully.SDS-PAGE test results showed that recombinant E.coli can effectively express the recombinant-toxin and two truncated-toxins by the inducer IPTG. Their expression productions were mainlyinclusion bodys, and a little of expression productions were soluble. The conditions of E.coli BL21(pET28a-csa) expressing recombinant-toxin were optimized. This test result showed the optimumconditions of inducing and expressing. The inducing time point was when the recombinant E.coli wascultured to the middle or end of logarithmic growth as numerical values of bacteria culture fluid OD600was in the range of0.850~0.950. The concentration of inducer IPTG was0.40mmol/L. The amount ofinducing time was for5~6h. And the inducing temperature was37℃. In this optimum condition, therelative expression quantity of recombinant-toxin could reach to25.0%. In the same conditions,recombinant E.coli BL21(pET28a219) could express34.0%for truncated-toxin CSAN219andrecombinant E.coli BL21(pET28a439) could express17.0%for truncated-toxin CSAC439.Biological characteristics of the recombinant-toxin and truncated-toxins were treated.Western-blot results showed that the recombinant-toxin and the truncated-toxins can react withantitoxin of Clostridium septicum specifically. The mouse toxicity test indicated that the recombinant-toxin had the lethal activity to mouse, which can be neutralized by the antitoxin of Clostridiumsepticum, but the truncated-toxins did not have lethal activity to mouse. And the cytotoxicity testshowed that the recombinant-toxin had cytotoxicity, but the truncated-toxins did not. And therecombinant-toxin and truncated-toxins did not have hemolytic activity on sheep blood.The rabbits were immunized with the vaccines of recombinant-toxin and truncated-toxins, and theblood of rabbits was collected after two weeks. The immune protection tests results showed that therecombinant-toxin had the best immunogenicity. And the recombinant-toxin of various expressingforms all showed higher immunogenicity, and could protect the immunized rabbits from challenge of-toxin of Clostridium septicum. The rabbits immunized with0.25mL of inactivated vaccine of recombinant-toxin (effective vaccine antigen content was about0.11mg) could be protected afterchallenged by-toxin of Clostridium septicum. However, the truncated-toxins had badimmunogenicity. And the rabbits immunized with1.0mL (effective vaccine antigen content were about1.05mg and0.58mg) of a kind of truncated-toxin inactivated vaccine could not be protected afterchallenged by-toxin of Clostridium septicum.The serum neutralization tests results were consistent with the immune protection tests ones. Theserum neutralization titers of protected rabbits were higher than that of unprotected rabbits. And theserum neutralization titers of rabbits immunized with recombinant-toxin bacterial vaccines weresimilar to the titers of rabbits immunized with ultrasonic disrupting bacterial vaccines, and the two titerswere higher than the titers of rabbits immunized with other expressing forms vaccines of recombinant-toxin, and the titers of rabbits immunized with vaccine prepared from the supernatant of ultrasonicrecombinant E.coli BL21(pET28a-csa) were same to the titers of rabbits immunized with vaccineprepared from the precipitation. And the test results of rabbits immunized with different immune dosesshowed that the neutralization titer of immunized rabbit serum increased as the increase of recombinant-toxin vaccine dose.And the immune protection rate can reach75%for sheep immunized whith0.50mL of the inactivatedbacterium vaccine of recombinant-toxin. To sum up, the recombinant-toxin of Clostridium septicumcan be used as a candidate material of preventive vaccine from infection of Clostridium septicum. |
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