|
In order to recognize the α toxin in the distribution of animals infected with Clostridium perfringens depth study of the pathogenic mechanism of the intestinal Clostridium perfringens source toxemia, this experiment homemade high titers of Clostridium perfringens α toxin polyclonal antibody as a primary antibody using immunohistochemical methods of artificially infected mice alpha toxin of Clostridium perfringens disease positioning clarifies the distribution of the toxin Clostridium perfringens reveal the pathogenic mechanism of the livestock sudden death syndrome. First incubated under anaerobic conditions for resurrection standard strains of type A Clostridium perfringens, and extract the DNA template, the design of primers specific for PCR amplification, cloning and its full-length gene of α toxin. Conventional recombinant techniques, the recovery of the target gene fragment inserted into the prokaryotic expression vector pET28a recombinant plasmid by restriction enzyme digestion, sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3), transformed completed after IPTG induction , groping induced optimal conditions, the the induction products under optimal conditions by SDS-PAGE and protein nickel column affinity purification purposes; PBS dialysis, Western blot detection specificity of the protein. Α-toxin protein to be expressed and purified 500μg mixed with immune Freund's adjuvant-immune rabbit four times, periodic serum antibody titers by ELISA method to obtain the polyclonal antibody. Toxigenic Clostridium perfringens inoculated medium, anaerobic environment 45C, 4h culture, take the culture liquid the conventional ammonium sulphate extraction exotoxin, diluted with sterile saline, abdominal subcutaneous injection mice taken within 72h of death small mouse organs and tissues: heart, liver, spleen, lung, kidney, stomach, small intestine, medulla, invested more than 4% paraformaldehyde solid-phase, and then making the tissue sections with homemade rabbit anti-alpha toxin polyclonal antibody as an anti- HRP (horseradish peroxidase) and FITC (fluorescein) labeled goat anti-rabbit IgG as secondary antibody. Mice suffering from Clostridium perfringens disease alpha toxin positioning detection by immunohistochemistry. The results showed that the PCR amplified product of 1228 bp in length, by Blast comparison, is recognized as the α toxin gene; the the expression vector pet28a and recombinant T vector containing the gene were digested, and the digestion line sexual pet28 recovery of the purpose of the product and digested fragments recovered product to connect successfully constructed pet28a-α expression vector. Induced by IPTG, the expression product obtained by SDS-PAGE electrophoresis, size 45KD expression of specific bands, the expression levels of up to 42.1%. Western blot results show that the expression of the target protein has a high degree of specificity. Expression of alpha-toxin protein antigen, four immunizing rabbits get much cloning antibody as measured by ELISA titer of 1: 512,000. Toxin distribution of organs by immunohistochemical method detection point out, the positive particles found in the cytoplasm of tissue cells in the medulla oblongata of mice, intestine, kidney, lung, stomach, and enter the description of the α toxin with the blood circulation system and infringes on animals medulla oblongata, intestine, kidney, lung, stomach, and other organizations. Positive particles were found in the blood vessels of the spleen, liver, the distribution toxin but not found in the main mass of cells, the toxin can not be against main interstitial cells of the spleen and liver. The study shows that the experiment is successful preparation of high titers of rabbit anti-alpha toxin polyclonal antibody immunohistochemistry results suggest for the first time in the α toxin found in the cytoplasm of the medulla oblongata, medulla oblongata is the center of life, respiratory, cardiovascular center toxins against it a disorder the combined toxins toxic effects on the digestive system, respiratory system and kidneys, resulting in the death of the animal.
|