| The pathogenicity of Clostridium perfringens(Cp)is related to a variety of toxins and extracellular enzymes produced by Clostridium perfringens.?2 toxin is one of them,and all types of Cp bacteria can produce it.More and more studies have shown that ? 2 toxin is also a very important toxin,but the toxic mechanism of ?2 toxin is not clear.Using monoclonal antibodies to detect antigens is a very important and convenient way.In order to explore the toxicity of ?2 toxin,the recombinant ?2 toxin protein was expressed and purified by E.coli expression system,and the monoclonal antibody against ?2 toxin was prepared by using the recombinant protein,and the effect of ?2 toxin was studied by in vivo rabbit ileal ligation,immunohistochemistry and porcine intestinal epithelial cell line(IPEC)in vitro.1.Vector construction and protein expression and purification: Using the JXJA17 strain preserved in this research group,based on the ?2 toxin and ? toxin genes it carrying,the recombinant plasmid(p ET-20b-?2,p GEX-4T-1 and p ET-20b-?)BL21 expressing bacteria,which realizes the large-scale expression of recombinant protein.Using the characteristics of His tag and Gst tag that can specifically bind to Ni and glutathione,respectively,relatively single recombinant proteins ?2-His,?2-Gst and ?-His were obtained.2.Animal immunity and antibody preparation and purification: BALB/c female mice were immunized with recombinant 32a-?2 protein,the ?2-Gst protein was used as the screening protein,and 3 hybridoma cells capable of stably secreting antibodies were successfully screened using hybridoma technology Strains(1B7,2C1,7D3).Using Western blotting to verify,the results show that 1B7,2C1 and 7D3 can specifically react with ?2-His,?2-Gst,and 32a-?2.The monoclonal antibody against ?2 toxin was successfully prepared.Kunming mice were immunized with recombinant protein ?-His as the antigen,and blood was collected by tail-cutting,and polyclonal antibodies against ?(immune mouse serum)were obtained.3.?2 toxin in vivo rabbit ileum ligation toxicity test: the ligated ileum of rabbits was injected with the bacterial supernatant treated with different antibodies for 6 hours.The results showed that the pathological conditions of intestine could not be judged well only from the eye view.The intestinal segment was sectioned and observed by HE staining.The results showed that ?2 toxin played a major role in the pathogenic process,which would cause the accumulation of red blood cells in the villi of the small intestine.The intestinal segment of the rabbit ileum ligation test was subjected to immunohistochemistry.The results showed that the ?2 toxin mainly aggregated on the surface of the intestinal villi and partly entered the lamina propria,and the ?2 toxin produced by different strains carrying the ?2 toxin gene was different.4.Study on the toxicity of ?2 toxin on porcine intestinal epithelial cells IPEC-J2 in vitro.Using bacterial liquid supernatants with different ?2 toxin content to act on IPEC-J2 cells,it was found that the bacterial liquid supernatant containing ?2 toxin would cause IPEC-J2 cells to round and lead to shedding.On the bacterial liquid neutralized by ?2 toxin antibody There is not much difference in the visual morphology between the clear-acting cells and the cells containing TGY medium and PBS.5.Yeast two-hybrid experiment:Used as a bait protein,?2 was fused with BD domain(DNA binding domain)of the Saccharomyces cerevisiae transcription factor GAL4 for expression.The c DNA library of porcine intestinal epithelial cell line IPEC-1 was constructed,then the c DNA library was fused with AD domain of GAL4 for expression.The proteins that exist in the pig intestinal epithelial cells and interact with ?2 toxin were screened and identified by yeast two-hybrid system.By yeast two-hybrid system,four potential proteins interacting with ?2 toxin were obtained,namely S20,LGALS1,L12 and Atrophin-1.Among these,LGALS1 is one kind of galectins that are located in the cell outer membrane and may be related to the invasion of toxins.Therefore,LGALS1 was verified by yeast retest analysis.The results showed that the interaction between C-terminal and ?2 toxin is stronger than that of the full-length protein.The results of this experiment will play an important role in elucidating the pathogenesis of ?2 toxin.In this experiment,the toxic effect of ?2 toxin was studied in vivo and in vitro,and the results showed that ?2 toxin played a major role in the pathogenesis of Cp A;?2 toxin mainly aggregates on the surface of intestinal villi,and part of ?2 toxin may interact with the galectin of intestinal epithelial cells.1 Combine,thereby entering the submucosa.This research lays a theoretical foundation for studying the pathogenic effect of ?2 toxin and helps prevent the pathogenicity of ?2 toxin. |
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