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Identification of the Differential Proteins in Small Intestinal between Piglets Susceptible and Resistant to ETEC F4ac

Author: ChenYiJie
Tutor: RenJun；HuangLuSheng
School: Jiangxi Agricultural University,
Course: Animal Genetic Breeding and Reproduction
Keywords: ETEC F4ac MUC13 gene Differences in protein Western blot Biotinylated
CLC: S828
Type: Master's thesis
Year: 2011
Downloads: 30
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Abstract

Porcine intestinal epithelial cells with or without the tools the adhesion activity the ETEC F4ac of receptor protein is to decide the Pig whether anti incidence of critical of ETEC F4ac infection. Early this laboratory through a series of genetic methods to porcine MUC13 gene identification for the purpose of encoding F4ac receptor gene, and found pig MUC13 gene the two Copy Type: MUCl3-A and the MUCl3-B. According MUCl3-A, and MUCl3-B deletion in intron 2 there is a 68 bp insertion mutation, a pair of primers designed at both sides and to identify both types of copy. The 286 Western pig detection found copies of MUCl3-A type homozygous pigs (100%) performance for ETEC F4ac resistance. These results show that of ETEC F4ac porcine intestinal adhesion depends on the variability of MUCl3-B. Tandem repeat region of the high GC content exists due the pig MUCl3-B copy coding region, it is difficult through the existing techniques of PCR and sequencing techniques to obtain its high GC content, the internal structure and sequence information of the tandem repeat region, such that in the level of genes and genomes. The MUC13 gene causal mutation studies difficult. Given in gene and gene set of technical limitations, this study is to from the protein level to start, the adhesion and not adhesion of the differences in characteristics ETEC F4ac and pig small intestine, the use of protein bio-biotinylated technology and Western blotting to identify their small intestine differences in protein, and then the use of mass spectrometry The technique to obtain the sequence information of the different proteins, in order to prove the gene encoding the difference for MUC13. First extraction and pili of purified F4ac bacteria, biotinylation of the fimbriae, and SDS-PAGE electrophoresis and Western blot identification the fimbriae whether successful biotinylation. Subsequently extracted to determine the the adhesion phenotype 6 the White Duroc intestine membrane protein, SDS-PAGE electrophoresis carried out in the same manner as, biotinylated F4ac pili as an anti do Western blot identification of differentially expressed proteins. The results show that 3 phenotype is a strong signal with the the White Duroc small intestine adhesion membrane protein 210 KDa and 240 KDa at three adhesion the White Duroc small intestine membrane protein 210 KDa and 240 KDa at None of the signal. 210 KDa and 240 KDa signal with corresponding SDS-PAGE strips with a knife cut, sending mass spectrometric identification. MS results than on MUC13 protein. The analysis may have two reasons: 1) the two proteins exist a lot of glycosylation; 2) Mass search existing protein database does not contain the MUCl3-B protein. This study have been identified to the ETEC F4ac adhesion phenotype between differences in protein, the center of gravity of the further work as the basis of the difference in protein deglycosylated to further reveal the amino acid sequence of the 210 KDa and 240 KDa ultimately confirmed This protein is encoded by MUC13 to provide the evidence of the direct protein levels.

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