| In order to understand the distribution ofα-toxin of animals infected Clostridium perfringens and deeply study the pathogenic mechanism of Clostridium perfringens intestinal toxemia, this experiment made the high price antibody of Clostridium perfringensα-toxin, and take the take the antibody as the first antibody to locateα-toxin in mice infected Clostridium perfringens by the method of immunohistochemistry to explain the distribution ofα-toxin and reveal the pathogenic of Sudden Death Syndrome.First of all, the A-type standard strains of Clostridium perfringens were cultured under anaerobic conditions,and then the extraction of DNA template,designation of specific primers, and the amplication and cloning of the length ofαtoxin gene by PCR. By the conventional recombinant DNA technology, the recovered gene fragment was inserted into expression vector pET28a to instruct the recombinant plasmid. By the enzyme digestion and the DNA sequencing, the recombinant plasmid was transformed into E.coli BL21 (DE3), and then was induced with IPTG to explore the optimal induction conditions. The product under the best conditions for the induction of SDS-PAGE electrophoresis was purified by nickel-affinity; PBS dialysis, Western blot detection of the protein specificity.The expressed and purifiedα-toxin protein 500μg was mixed with Freund adjuvant to immune rabbits four times. The serum was regularly checked to detect antibody titer by ELISA to obtain polyclonal antibodies.Clostridium perfringens were inoculated in toxin production medium, anaerobic environment, 45℃, 4h, and then the culture medium was obtained exotoxin by conventional extraction of ammonium sulfate and then was diluted with sterile saline, and was intraperitoneally injected mice. Take the small mouse organs died within 72h: heart, liver, spleen, lung, kidney, stomach, small intestine, medulla, into 4% paraformaldehyde, and then make tissue sections, using home-made rabbit polyclonal antibody againstα-toxin as first antibody,and the goat anti-rabbit IgG antibody which were labeled with HRP(horseradish peroxidase) and FITC (fluorescein) as secondary antibody. By immunohistochemistry mice suffering from Clostridium perfringensα-toxin were positioning detected.The results showed that: 1228bp length product was amplified by PCR and was confirmed indeed theα-toxin gene by Blast comparison; pet28a-αexpression vector was constructed successfully after the expression vector and the target gene were double digested and then connected; a specific band with molecular weight of 45kDa was appeared after the SDS-PAGE electrophoresis of expression products; Western blot results proved that the expressed protein as antigen obtained had a high degree of specificity; the titer of polyclonal antibody above was 1:512,000 after four times immunization of rabbits.Rabbits were immunized four times with theα-toxin protein as antigen. The antibody was obtained and was tested by ELISA which showed the result the titer of 1: 512,000. cloning is much more,.By the immunohistochemical detection, positive particles were found in the medulla oblongata, intestine, kidney, lung, stomach tissue cells in the cytoplasm, indicating thatα-toxin violated those organs with the blood circulation system. In the spleen, liver positive particles were found in blood vessels, but in the main cells, indicating that toxin can not be against the main mass cells of liver and spleen. Studies have shown that, the success of this experiment was the preparation of high titer rabbit polyclonal antibody againstαtoxin. Immunohistochemistry results suggested that,α-toxin was found in the medulla oblongata for the first time. Medulla oblongata was the hub of life, respiratory, cardiovascular center, for the reason of toxins against their disorders, in addition to the toxicity to the digestive system and kidney, animal death finally. |
