Construction Of Mutants Of Clostridium Perfringens Alpha,Epsilon Toxins And Its Application

Clostridium perfringens is divided into five toxinotypes (A-E) according to the four main lethal toxins (alpha, beta, epsilon and iota toxins) produced by these strains. Alpha-toxin (a-toxin), produced by all types of C.perfringens, could cause gas gangrene. α-toxin has the biological activities of phospholipase C (PLC), sphingomyelinase, hemolysis and lethality. Epsilon-toxin (ε-toxin), which is produced by types B and D strains, is considered a potential biological weapon.ε-toxin can lead to fatal illnesses in livestock animals, especially induce enterotoxemia in sheep. These toxins pose major biological weapon or bioterrorism threats, and have been listed for Export Control of Dual-Use Biological Agents. Consequently, it is necessary to develop the antibody or vaccine against these toxins to protect human or animals.In this study, we constructed8mutants of the two toxins and transformed them into E. coli for expression and purification, the antigenicity and biological activity of mutants were detected by a series of tests. The mutants which has low or non-toxicity and retained the immunogenicity were selected to be the candidate vaccine or be prepared to produce IgY.Firstly, the recombinant plasmid pTIG-ETX used as the template, the six mutants pTIG-mETXH106P, pTIG-mETXS111H, pTIG-mETXS111Y, pTIG-mETXF199H, pTIG-mETXF199E, pTIG-mETXS111YF199E were generated with the site-directed mutagenesis. Then the mutants were transformed into E.coli-competent BL21(DE3), the mutants sequence were verified using nucleotide sequence analysis. The BL21(DE3)/mETXs were grown at37℃and induced with0.5mM IPTG at a temperature of16℃overnight, then the bacteria were collected and the lysate was purified through HiTrap Chelating high performance column with Ni2+. After the antigenicity of mETXs were identified, the cytotoxicity of mETXs were measured and the dose of mETX required to kill50%of the MDCK cells was calculated and compared with that of rETX. Balb/C mice were immunized with mETXH106P or mETXF199E, the sera of immunized mice were titrated using an indirect ELISA. One week after the last immunization, the mice were challenged with active rETX and the survival status would be observed for72h.Secondly, two mutants pTIG-mCPAD56S and pTIG-mCPAH68s were generated with the site-directed mutagenesis and were transformed into E.coli-competent Origami for expression. The nucleotide sequences of mCPAs were confirmed by sequencing. The Origami/mCPAs were grown at37℃and induced with0.5mM IPTG at a temperature of16℃overnight, then the resulting proteins were purified using Ni2+-chelating chromatography. After test the immunogenicity and the bioactivity, two health hens were selected and immunized with mCPAs, harvested eggs after7days of the first immune. IgY was obtained by the water dilution method and the titers were detected by indirect ELIS A. The polyclonal antibody were stored by lyophilization.Finally, a group of mETXs and mCPAs have been constructed and purified, a low toxic mETX has been found to be the candidate vaccine against poisoning by ε-toxin, and IgY against a-toxin which could be used for detecting a-toxin were obtained for the further work.