Study On The Mechanism Of MUC13 Gene Against Pathogenic Escherichia Coli

Enterotoxigenic Escherichia coli(ETEC F4ac)is one of the major pathogens in pig.Enterotoxins produced by ETEC can cause diarrhea in piglets.The F4 pilin protein of ETEC binds to the piglet's intestine receptor protein and assists enterotoxins into the small intestine epithelial cells and causes diarrhea.If the small intestine encodes a receptor protein,the piglets might occer diarrhea,individuals that do not express pilin protein will not develop diarrhea even if they are infected by ETEC.Therefore,the pilin protein receptor genes determine whether piglets are susceptible to ETEC.The MUC13 gene located at chromosome13 is of pig considered as a receptor gene for controlling susceptibility or resistance of piglet ETECF4ac~+.This article focuses on the detection of the SNP sites of MUC13 intron 7 and all exons.Allele-specific PCR method was used to study the polymorphism of MUC13 gene in Guizhou local pigs.Site-directed mutagenesis was used to link the coding regions of the two genotypes to the Saccharomyces cerevisiae expression vector pYES3-CT,the recombinant plasmid pYES3-MUC13 was constructed and induced to express two type of proteins.Flow cytometry was used to test the binding ability of the two types proteins to ETEC F4ac~+strain,the following results have been obtained:1.The detection of a known mutation site in intron 7 of MUC13 gene showed that frequency of G gene in Guizhou local pigs was significantly higher than that in Large White pigs,and the difference was extremely significant.2.All of exons of the MUC13 gene were sequenced and a total of five candidate SNPs were detected,located in exon 6,exon 7,and exon 8,respectively.Only the mutation site C-G in exon 6 and the mutation site A-G in exon 8 were the missense mutation with threonine/serine,glutamine/arginine substitution.The remaining 3 sites belong to synonymous mutations and did result in amino acid changes.3.The linkage analysis between the MUC13 intron 7 mutation site and the mutation site in exon 6 revealed a low degree of linkage between them.4.Adhesion experiments with different genotypes of piglet intestinal cells and enterotoxigenic Escherichia coli confirmed that pig intestine cells on 6 of MUC13 protein with GG-type in exon 6 could adherent to strain,CCporcine intestinal cells and ETEC F4ac are weakly adherent.5.The coding region of MUC13 gene was amplificated by RT-PCR,and two types of MUC13 gene coding regions were inserted into the eukaryotic expression vector pYES3-CT by site-directed mutagenesis method,the electroporation transforms into the yeast strain INVSc1 and induced by galactose.Flow cytometry was used to detect the binding ability of both types of proteins to ETEC F4ac~+strain.In summary,the weakly-adherent genotypes of MUC13 gene predominated in the local pig population in Guizhou predominated.Two of yeast MUC13 proteins were heterologously expressed using the yeast expression system.In vitro experiments confirmed that the G/C mutation in exon 6 of the receptor gene MUC13 changed the binding ability of the protein to ETEC F4ac~+.The results can lay a theoretical foundation for the breeding of anti-diarrhea pig breeds.