Studies On Pilin, Cluture And Extraction Of Pili Among Several Strains From Porcine Enteropathogenic E.coli

Escherichia coli is one of the most important pathogens in young animals. Infection ofenterotoxigentic Escherichia coli(ETEC) is a major factor of causing diarrhea and death inweaned piglets. The pathogenesis of ETEC is strongly related to adhesive pili andent-erotoxins. Pili are hair-like proteinaceous appendages, playing an essential role in thepathogenesis of E.coli infection by mediating adhesion to epithelial cells when theadhesions on the surface of bacteria bind to hosts cells so that the bacteria can localize inlocal tissue and produce toxin or destroy tissue, leading to diarrhea.In this thesis, 12 strains among Porcine E.coli, isolated from diarrhea piglets,carryingon the biochemical identification and the pathogenic examination, Screening out of theseven have pathogenic E. coll. Byelectron microscopy revealed EC308, EC316,EC358and EC369 four E. coli cultured in TSB medium, 37℃for 48h, passaged threeconsecutive times pili can be a good expression. The test proved that heat treatment,ammonium sulfate precipitation method, which can effectively guarantee pilin extractionvolume and purity. Used four isolated strains Preparation of bacteria antiserum and piliantiserum.The E.coli antigens serological cross-test indicated that the four of E. coliantigen and pili antigen cross each other, Pili is the major protective antigen in the fourstrains of Escherichia coli.Extraction of pilin by SDS-PAGE detected the protein molecular weight, there werethree protein molecular weight in EC369 and EC358 pili extracts,which is20.0KD,40.5KD,72.0KD and 20.0KD,36.4KD,85.6KD; EC316 pili extract have four of theprotein bands with molecular weight of 20.0KD,38.4KD,45.0KD and 66.2KD; EC308 pili extract only 24.0KD a molecular weight of the protein bands. But EC369, EC358 andEC316 contain the same molecular weight of the protein bands 20.0KD.Western-blotting experiments showed that 20KD molecular weight of protein bandsof EC369,EC358,EC316 and the molecular weight of the EC308 24KD protein bands havereaction bands with pili antiserum of the 4 strains.Therefore, the combination of mannose-sensitive hemagglutinin test (MRHA) resultscan be judged EC308 expression of pili as K88, EC369, EC358 and EC316 expression ofpili as 987P. The results indicated that the four different pilin of E. coli have commonantigen epitope.