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Subject: Animal Biotechnology × Author: Bharali, Arpita × Start date: 2000 ×
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    ThesisItemOpen Access
    Production and characterization of recombinant beta toxin of Clostridium perfringens
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-03) Bharali, Arpita; Sharma, R K
    Clostridium perfringens causes several forms of enteric disease in human and animals. Different toxinotypes of C. perfringens produce different combinations of lethal toxins. C. perfringens type C produces beta toxin which is a kind of lethal pore-forming toxin that is responsible for necrotic enteritis in animals. Although C. perfringens type C infects several livestock species, the juvenile pigs are most susceptible. In India, the north-eastern states have the highest pig population in the country but no indigenous vaccine against C. perfringens type C is currently available. Therefore, the present study was conducted with an aim to produce recombinant beta toxin protein in a heterologous host and to evaluate the immunogenicity of the recombinant toxin adjuvanted with calcium phosphate nanoparticles in the mice model. For this, the cpb gene of C. perfringens type C that encodes the beta toxin was cloned into a prokaryotic expression vector, pET28a(+). Then the recombinant clone was transformed into BL21-CodonPlus®(DE3)-RIL E. coli cells. Expression of the recombinant beta toxin was induced by 1 mM IPTG for 12 hours at 37C. The recombinant beta toxin protein was present as inclusion bodies in the insoluble fraction of the cell lysate which was further purified by Ni-NTA column affinity chromatography and confirmed by SDS-PAGE analysis. The specificity and the reactivity of the recombinant beta toxin protein were confirmed by western blotting using anti-sheep C. perfringens beta toxin serum. In-vitro and in-vivo toxicity of the recombinant beta toxin protein was evaluated in MDCK cell line and mice, respectively. The recombinant beta toxin protein did not show cytotoxicity in the concentrations from 3500-6.89 ng/ml as well as failed to produce clinical signs or death in mice when administered intravenously at a 100μg dose. The recombinant beta toxin protein was loaded into calcium phosphate nanoparticles (CaP-NPs) used as an adjuvant, and a calcium phosphate nanoparticles-recombinant beta toxin protein complex was produced which was characterized using a zetasizer. The immunogenicity of the recombinant beta toxin protein adjuvanted with CaP-NPs was evaluated and compared with the conventional Freund’s adjuvant in the mice model. Three groups of six mice each were inoculated separately with 30 μg of the recombinant beta toxin protein with Freund’s adjuvant (Group-I), 30 μg of the recombinant beta toxin adjuvanted with CaP-NPs (Group-II), and PBS in the control group (Group-III). The booster doses with corresponding inocula were given in all the groups 3 weeks apart and the level of antibody was estimated in serum samples collected at 7 days interval from day 0 to day 35 post-primary inoculation by indirect ELISA. The specific antibody response against the recombinant toxin protein was significantly higher (p<0.05) in Groups I and II from day 7 to day 35 compared to the control group. The level of antibody was at its peak on day 28 in the experimental groups (Groups I and II) and from day 28 to day 35, the level of antibody was significantly higher (p<0.05) in Group-II compared to Group-I. Hence, in the present study a non-toxic form of recombinant beta toxin of C. perfringens could be expressed in E. coli and the combination of the recombinant beta toxin protein with CaP-NPs as adjuvant could elicit better antibody response compared to its combination with the conventional oil adjuvant. However, its ability to produce neutralizing antitoxin and the protective efficacy against challenge infection are to be ascertained in future for considering it as a potential vaccine candidate against C. perfringens type C infection.