Identification Of The Differential Proteins In Small Intestinal Between Piglets Susceptible And Resistant To ETEC F4ac

The pathogenicity of ETEC F4ac is mainly determined by its colonization to the specific receptor on the brush border of villous entrecotes in the small intestine. Previously, our lab has identified MUC13 as the gene coding the receptor for ETEC F4ac in the porcine small intestine, and revealed that the MUC13 has two copies:MUC13-A and MUC13-B, which have been distinguished by an InDel of 68 bp in intron 2. In this study,286 pigs of 15 Chinese native breeds and Western breeds were tested. The results showed that all pigs homozygous for MUC13-A (n=38) were resistant to ETEC F4ac, indicating that the adhension between ETEC F4ac and the receptor is solely determined by variation in MUC13-B, rather than MUC13-A.Both MUC13 copies contain tandem repeat regions with rich GC content in coding regions. So it's difficult to determine the complete sequence of the repeat region of MUC13 by using the current available PCR or sequencing technology. We thus try to identify the differential proteins interacting with ETEC F4ac though protein techniques.First, we extracted and purified the F4ac pili; then biotinylated the F4ac pili proteins that were detected by SDS-PAGE and Western Blot. Second, we obtained the membrane proteins in small intestine of six White Duroc pigs with adhesive or non-adhesive phenotypes. Then, the membrane proteins were separated by SDS-PAGE and detected by Western Blot using biotinylated F4ac pili as the first antibody. The results revealed that the adhesive piglets had intense signals at positions 210 KDa and 240 KDa, and the non-adhesive piglets had weak signals in the locations. We further isolated the differential proteins that were then subjected to mass spectrometry assay. However, the results did not contain the MUC13 peptide. It could be due to two reasons:1) Rich glycosylation in the differential proteins.2) The protein database does not include the MUC13-B protein. The following work will focus on dealing with glycosylation to clearly reveal the differetial protein and demonstrate the previous conclusion that the ETEC F4ac receptor is encoded by the MUC13 gene.