Basic Studies On Non-Toxic Mutant Vaccine Of Clostridium Perfringens Epsilon Toxin(ETX)

Clostridium perfringens,widely distributed in nature and intestines, is one of the important zoonotic pathogens. Clostridium perfringens produced many exotoxin, according to the main four lethal toxins α、β、ε and ι, Clostridium perfringens is divided into five toxinotypes, namely B、C、D and E. Epsilon toxin, produced by toxinotypes B and D, is a severe pathogenic factor, epsilon toxin is responsible for fatal enterotoxemia in sheep、goat and other animals. Enterotoxemia can make animal die in a short time and antibiotic treatment dose not make sense, that causes serious economic losses to the farming industry worldwide. The best way to prevent the fatal enterotoxemia is to inject vaccine. Compared with traditional toxoid vaccines, researches about recombinant epsilon toxin vaccine are becoming more popular.In this study, we screened the non-toxic or low toxicity mutants by changing the construction and substituting of Y196 with glutamic acid, the plasmids pET-His-etx and pTIG-etx which constructed by our lab were used as a template,Glu mutant was introduced into plasmids by Polymerase Chain Reaction(PCR) according to the instruction of QuikChange Lightning Site-Directed Mutagenesis Kit(Stratagene, Beijing, China), then the plasmids were digested by Dpn I enzyme and the accurate plasmids were transformed into competent Escherichia coli BL21(DE3)pLysS cells(Transgen, Beijing, China). We transferred monoclonal colony into 5 mL of LB culture and sequenced the DNA of Escherichia coli, the correct bacterial were added to 500 m L of LB culture according to the scale of 1:100, the cells were cultured at 37℃ to an A600 of 0.6-0.8, then the IPTG added to flask and cultured at 16 ℃. The cells were centrifuged at 8000 r/min, for 15 min, retained the precipitate, and washed the precipitate with PBS, and then the precipitate was washed with buffer A and lysed by sonication. The supernatant was collected by centrifuging and purified with a precharged Ni2+ column. Proteins were evaluated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The purified proteins were incubated with MDCK cells and injected into mice, the value of CT50 and LD50, were calculated by MTS and the improved Karber’s method, which were used to evaluate the effect of the purified proteins.To clarify the mechanism of proteins to lose toxicity, we selected the low-toxic mutant proteins to further research. First, the MDCK cells were used to check the binding of mutant, the cells were incubated with different concentration of toxins and then incubated with 4% formaldehyde for 15 min, after incubated with 5% BSA, the cells were incubated with anti-His antibodies and secondary antibodies. The signal was detected using multifunctional plate reader and confocal microscope. The western-blot was used to check the heteromeric polymerization. Then using a calcium ion probe to detect calcium, the MDCK cells were incubated with probe, after washing with PBS, the mutants were added, the fluorescence signal at 190-260 nm which reflect the ability of pore formation was detected by multifunctional plate reader and the PBS used as a background.The epsilon toxin mutant rETXY196E-C, one of the four mutants, was selected as the antigen to immune mice. To optimize the immune methods, the Babl/c mice were divided into 7 groups randomly, 15 mice in each groups, there are three concentration of rETXY196E-C and two immune approaches in this study, it was 5 μg/mouse subcutaneous, 5 μg/mouse intraperitoneal, 10 μg/mouse subcutaneous, 10 μg/mouse intraperitoneal, 15 μg/mouse subcutaneous, 15 μg/mouse intraperitoneal and 0.01 M PBS. The mice were immunized three times and weight changes were recorded, serum, collected from mice one week after each immunization, was used to measure the antibody titers, after third immunized, a part of mice were used to challenge experiment, mice in protein immunized groups were challenged with 100 × LD50, 500 × LD50 and 1000 × LD50 dose of wild toxin, however, mice in PBS immunized groups were challenged with 10 × LD50 dose of wild toxin. Another part of mice were killed to separate the spleen, after lysing, the IL-4 and IFN-γ cytokines in T lymphocytes were detected by flow cytometry. To detect the neutralisation of antibody, the serum mixed with equal volume of r ETX at 37℃ for 30 min and the mixture was incubated with MDCK cells and to inject mice. To detect the antibody affinity between sera and rETX or rETXY196E-C, the proteins were coated into 96-well plate, and then the sera and anti-ETX antibody were added to the 96-well plate, wash the plate with different concentration of NH4 SCN to test the affinity index.In this study, four epsilon toxin mutants rETX 、 rETX-C 、 rETXY196 E and rETXY196E-C were expressed, and suggested that the amino acid residue Y196 E substitution and the C-terminal peptide synergistically alleviate ETX toxicity, the C-terminal peptides mainly inhibit the binding activity and the Y196 E substitution slightly inhibits heptamer-forming process. The results of animal and cell experiments showed that The LD50 of rETX-C and rETXY196 E was 110, 000 and the LD50 of rETX was 7910, however, the LD50 of rETXY196E-C was 1770, 000. Those results showed that the toxicity of ETX reduced the same level when only to substitute the Y196 amino acid or introduce the C-terminal to ETX. The substitution of Y196 reduced the toxicity of ETX by an unknown mechanism.The results of animal experiment showed that protein rETXY196E-C could stimulate the immune system of mouse and make it produce lots of neutralizing antibodies. Among the six groups, 80% mice in 15 μg/mouse subcutaneous group survived at 1000 × LD50 of rETX and 100% mice survived at 500 × LD50 of rETX, however, mice in intraperitoneal groups only survived at 100 × LD50 of rETX. The results of histopathologic experiment showed that after challenged with dose of 1000 × LD50 rETX, brain of mice in PBS immunized group showed cytoplasmic vacuolation and kidneys showed obvious pathological alterations, including serious hemorrhage and mild edema when compared to that of control group. However, the mice symptom was significantly improved in rETXY196E-C immunized group. Those results showed that the mice immunized with recombinant protein rETXY196E-C could protect their sensitive organ from damage by r ETX. Those results suggested that immunize mice subcutaneously better than intraperitoneally. The results of antibody affinity showed that the antibody affinity between antibody and rETXY196E-C or rETX had no signifiacant difference. Therefore, the way to screen epsilon toxin mutant vaccine by changing structure as well as substituting amino acid was feasible, and protein rETXY196E-C is safe and contains immunogenicity, protein rETXY196E-C can be considered as a potential vaccine candidate for future researches.