| Escherichia coli isolated strain GL7 (078), was inactivated as immunogen to prepare specific monoclonal antibody. 4 strains of monoclonal antibody hybridoma cells were obtained by indirectly enzyme-linked immunosorbent assay (ELISA) and slide direct agglutination (DA). They are all IgG, all the 4 strains were specific to the type 1 pili through Western-blotting, and recognized the protein of fimbriae which MW about 17-17.5kd. There antibodies were used as probe to cheek the type 1 from Escherichia coli isolated strain from chicken, the result showed the there were exist differences whether there were in the same serotype or not, and 0 serotype can't fully embody the hereditary relativity of different strains.According to the published type 1 pilin pilA nucleotide sequence from avian Escherichia coli, a pair of primers were designed and synthesized, which localize conservative regions of type 1 structural gene. 26 positive 657bp products were generated by polymerse chain reaction (PCR) from 20 different serotypes avian Escherichia coli pathogenic isolates' chromosomal samples. Direct PCR amplification of cloned fragment and enzyme digest indicated that all 26 DNA fragments had been cloned into pGEM4-plasmid vector successfully, and nucleotide sequences of all positive DNA fragments were analysed. With the help of DNA-Star software, 26 pilA gene nulceotide sequences from 26 avian Escherichia coli were compared, 3 different types 1 pili genotypes were found among 26 avian Escherichia coli strains. Theywere named 1A, 1B and 1C, respectively. Among 26 avian Eschierichia coli strains, the percentage of 1A, IB and 1C are 23.1%(6/26), 26.9%(7/26) and 50%( 13/26), respectively; the similarity of amino acid sequences from 1A, IB and 1C are 91.35%-92.36%. The genotype of type 1 pilin hasn't relateness to serotype of them.Three isolating strains from chicken were used for strains of making monovalent and multivalent pili oil-emulsified inactivated vaccine, it indicated by the experiment that type 1 pili vaccine have cross-protection immunizing effect to different serotype Escherichia coli.4-week-old chickens were vaccinated with monovlent type 1 pili vaccine, suffered 0-8.33% mortality after challenged with homologous pathogenic Escherichia coli, the mortality was 33.3%-58.3% after challenged with heterologous pathogenic Escherichia co//,but unvaccined chickens suffered 75%-91.67% mortality after with pathogenic Escherichia coli strains.Chickens vaccined with multivalent type 1 pili oil-emulsified vaccine suffered 13.3% mortality after challenged with homologous pathogenic Escherichia coli(011,018 and O78),unvaccined chickens suffered 70% mortality.Vaccined chickeds suffered 3.33% mortality after challenged with Escherichia coli 02,its negative control group suffered 61.53%mortality.It suggested that the vaccine not only have good immunizing effect against homologous Escherichia co//,but also have some cross-protection effect against heterologous Escherichia coli.According to the published type 1 pilA nucleotide sequences from avian Escherichia coli, a pair of primers were designed and synthesized, which localize conservative regions of type 1 pili pilA gene and were modified with BamH I and Sal I enzyme site. A 551bp DNA fragment was generated by PCR from chromosomal samples of Escherichia coli strain GL7 (078). Via the digestion of Sal I and BamH I and Linkage , the pilk gene removed from the 551 bp DNA fragment was cloned into the plasmid vectorpET-32a(+) .The recombinant expression plasmid was constructed and named F15pilA/pET-32a(+), and was transferred into Escherichia coli BL21.The recombinant bacterium BL21 (DE3- F15pilA/pET-32a( + )) was generated by screen. The results of SDS-PAGE and Western blot demonstrated that recombinant fusion protein was expressed by F15pilA/pET-32a(+) plasmid. The puried recombinant protein oil-emulsified vaccine was prepared to evalute its immunogenicity, the vaccine contained 400ug/ml recombinant protein. 4-week-old chickens were vaccinated subcutaneously and challengd via the posterior thor... |
PDF Full Text Request