Study On The Toxic Effects Of Salmonella Typhimurium Stn Toxin Of Porcine Origin And Screening Of Antagonistic Lactic Acid Bacilli

Salmonella typhimurium is one of the most prevalent Salmonella serotypes globally,with a wide host range and high pathogenicity,and is closely associated with livestock development and human health.The pathogenic process of Salmonella typhimurium is the result of a combination of pathogenic factors.The stn toxin,encoded by the stn gene,was first identified in Salmonella typhimurium,a virulent protein that causes increased fluid secretion in the intestinal tract of animals.Some studies have shown that its presence is essential for the full pathogenicity of Salmonella.However,the mechanism of enterotoxin action is not well explained.In this study,a strain of Salmonella typhimurium carrying the stn gene was isolated,we successfully constructed the pET-32(+)-stn plasmid to achieve in vitro expression of stn toxin and obtained an anti-stn toxin polyclonal antibody.To investigate the mechanism of toxic action of stn toxin by pull-down combined mass spectrometry and pig small intestinal epithelial cell line IPEC-J2 attack assay.We screened for Lactobacillus antagonistic to Salmonella typhimurium by in vitro inhibition tests,gentamicin protection and animal testing.1.Bacterial isolation: A strain of Salmonella typhimurium was isolated from the intestinal tract of dead weaned piglets.The pathogenicity of the bacterium was established in animal tests,and PCR detected that the bacterium carried the inv A,stn and sop B genes.The composition and differences of the intestinal flora of Salmonella-infected piglets and healthy piglets in the same group were analysed by bacterial 16 S r DNA high-throughput sequencing.The result showed that the abundance of Proteobacteria and Actinobacteriota was higher in the glandular diarrhoea piglet group than in the healthy piglet group,while the abundance of Enterorhabdus,Acidaminococcus and Sphaerochaeta was significantly lower in the genus horizontal diarrhoea group than in the healthy group.The percentage of Salmonella spp.was higher in the diarrhoea group than in the healthy group.2.Expression purification of recombinant stn toxin and preparation of polyclonal antibodies: The pET-32a(+)-stn plasmid was constructed and Ni columns were used to purify the recombinant protein.Cell attack assay shows that recombinant stn toxin can cause IPEC-J2 cells to round up,shed and die.The recombinant stn toxin was used as the immunogen for multiple immunizations of 6-week-old BALB/c mice,and the sera were obtained as anti-stn toxin polyclonal antibodies.This was validated by Western blot and the results showed that a polyclonal antibody against stn toxin with good immunogenicity and specificity was successfully obtained.3.Screening and identification of interacting proteins between recombinant stn toxin and IPEC-J2 cell: We screened IPEC-J2 cells for interacting proteins with recombinant stn toxin using the Pull-down method and analysed the differential bands in the pET-32a(+)proteome versus the pET-32a(+)-stn proteome,identified using mass spectrometry.Proteins with high confidence in the mass spectrometry results are those that may interact with stn toxins,including myosins,keratins,microtubulin and wave proteins,which form the cytoskeleton;various ribosomal proteins;lattice proteins involved in material transport;IF-like rod-like structural proteins and elongation factors involved in protein synthesis;histone like proteins involved in DNA replication and repair;functional fusion proteins;nucleophosmin and membrane linked proteins.4 Screening of probiotic lactic acid bacteria antagonistic to Salmonella: In this study,two strains of porcine-derived Lactobacillus spp.were screened for significant growth inhibition of Salmonella typhimurium by in vitro inhibition tests.Cellular assays showed that both strains of Lactobacillus significantly reduced the viability and ability of Salmonella typhimurium to invade IPEC-J2 cells,with Lactobacillus salivarius(H1)being the more potent inhibitor.The results of the animal tests showed that the mortality rate of mice in both the Lactobacillus salivarius prophylactic and treatment groups was significantly reduced compared to the Salmonella attack group,with no mortality in the prophylactic group and the high dose of Lactobacillus showed better protection against Salmonella infection.H1 had a better effect against Salmonella infection.In conclusion,this study provides a theoretical basis for the study of the mechanism of Salmonella stn toxin through the screening and identification of recombinant stn toxin and IPEC-J2 cell interaction proteins and the attack assay on IPEC-J2 cells.The screening of probiotic lactic acid bacteria,which are effective in reducing Salmonella infestation in in vitro and in vivo experiments,has laid the theoretical foundation for the development of alternative anti-drugs.