High Expression And Immunogenicity Evaluation Of Alphatoxin, Beta-toxin And Epsilon-toxin Of Clostridium Perfringens C58-1 Strain And Improvement Of Manufacturing Technique

The strain, Clostridium perfringens, also called Cl.wechii causes human gas gangrene, and enterotoxaemia and necrotic enteritis in animals, which is the key pathogen for sudden death syndrome of live stocks in China. The previous report implies that clostridium toxin is an effective vaccine antigen and the inactivated toxin produced during the clostridium cultivation can provide a good immunity against clostridiosis. The inactivated vaccine containing ovine/caprine braxy, strick, lamb dysentery and enterotoxaemia is proved to be highly protection and widely used in China.Currently, the inactivated vaccine is usually manufactured by anaerobic clostridium with the meat soup digested by anaerobic liver enzyme, and the medium with too many raw materials, such as livers, beef and enzyme contributes to the high cost during the vaccine production. Moreover, the quality control of the above raw materials(i.e. fresh meat, antibiotic residues and etc) is difficult to control and it will affect the stability of the final products. Furthermore, the other disadvantages are large doses with severe side effect post immunization. In current study, four toxin proteins of C58-1(α, β1, ε and β1-ε) were highly expressed and identified. Both mice and rabbit were inoculated with the above toxin proteins and then challenged with the toxins to determine the efficacy. Finally, the optimization of the protein expression was carried out to get the vaccine antigens.Consequently, the four toxin proteins, α, β1, ε and β1-ε of C58-1 were cloned successfully, and the expression vectors, p ETα765, p ETβ1927, p ETε984 and p ET1947 was constructed and transferred into E.coli BL21 to express the target proteins. Four target proteins were confirmed to have good immunoreactivity with the positive antibodies using Western blotting assay.Mean while, sequences of α, β1, ε and β1-ε of C58-1 were analyzed using genomic software. The results displayed that α toxin protein shared 61%-100% similarity with the protein of C. perfringens type A-E, α toxin of C58-1 strain shared the lowest homology(61%) while type C had 98%-100% similarity as compared to type B-E strain. More important, the α toxin was indentified to have a similar function in comparison with C. perfringens type B-E strain. In other hand, β1 toxin has 99.4%-100% homology in comparison with C. perfringens B and type C. ε recombinant protein of C58-1 showed 99.7%-100% similarity with C. perfringens B and D strain and it might have the same biological function as C. perfringens B and D strain shared.Post immunization with above 4 recombinant toxins, mice with the recombinant α or β1, could not get full protection after challenge. On contrary, mice inoculated with ε or β1-ε were well protected and their immunity were superior to whole bacterial vaccine. Particularly, β1-ε toxin was confirmed to provide 100% protection against C. perfringens B, C or D infection in mice model. According to the production and examination principle of inactivated combined ovine/caprine braxy, struck, lamb dysentery and enterotoxaemia vaccine in PRC veterinary Pharmacopoeia, the mothod of serum neutralization was used to determine the β1-ε toxin vaccine and the results indicated that β1-ε toxin vaccine accorded with national standard.With respect to mechanism of acetate synthesis and recombinant protein expression, the highly expression of β1-ε toxin in E.coli can be achieved by reduction of acetate accumulation. The effects of p H and the dissolved oxygen(DO) on expression of β1-ε toxin were optimized. The results indicated that highly cell density, large quantity of β1-ε toxin and low accumulation of acetate were achieved by controlling the following references, such as p H at 6.5(0-6 h), 7.0(6-16 h) and DO levels at 60%(0-6 h) and 30%(6-16 h). Furthermore, the effect of intermittent medium supply, DO feedback, p H feedback and glucose supply on β1-ε toxin expression were investigated, the optimal references showed that the cell density reached 2.045(absorbance at 600 nm) and the recombinant proteins were 63.24 mg/L while the accumulation of acetate decreased to 0.872 g/L using the combination approaches of p H control and DO in different phases as well as DO feedback method. In this way, the optimization β1-ε toxin protein was achieved by the selection of the fermentation references and procedures by decreasing acetate accumulation.