Cloning, Expression And Biological Characterizations Of Virulent-Related Gene Fragment E9 Of Avian Pathogenic Escherichia Coli IMT5155

Avian colibacillosis caused by pathogenic Escherichia coli is an acute or recurrent infective desease of avian. Predominant serotypes of avian pathogenic E.coli(APEC) are O1:K1,O2:K1 and O78:K80. Various virulence factors of APEC strains, such as lipopolysaccharide complexes, capsule, adhesins, temperature-sensitive hemagglutinin, iron acquisition systems, outer membrane proteins, toxins, AGI-3 genome island, ibeA, Pst system and colicin have been reported. The virulent gene fragments E9 and F11 were the two of the fragments obtained by SSH between avian high pathogenic E.coli EMT5155 with avian nonpathogenic E.coli CFT073. In this research the two fragments were cloned and sequenced, and E9 fragment was expressed and biological characterizations of the expressed protein were analyzed.1. Based on the sequence of E.coli published on the GenBank after BLAST, ORFs of gene fragments E9 and F11 were amplified from genomic DNA of APEC IMT5155 strain by polymerase chain reaction (PCR).Then the PCR products were inserted into pMD18-T Simple Vector routinely, thus constructing the cloning vectors pMD-E9/F11. The lengths of the cloning fragments E9 and F11 were 750bp and 1917bp, encoding 250 and 639 amino acids, respectively. Compared to that of other genetypes, they showed identity of 100% and 99.9% to that of APEC 01 respectively. The results of DNAStar Protean analysis indicated that virulence gene E9 and F11 fragments of IMT5155 strain shared similar antigenicity with other strains of E.coli.2. Digested with EcoR I and Xho I, a 750bp of the PCR product was cloned into the expression plasmid vector pET-28a (+). The recombinant was transformed into the BL21 and induced to express by 1.0mM IPTG at 37℃.The expression product was identified by SDS-PAGE and a band with 32.2kD as expected was obtained. Western blotting revealed that the recombinant protein was reactive with rabbit serum against E.coli IMT5155 suggesting that the protein is immunogenic.3. The protein with a single band of 32.2kD was obtained by HisTrap^TM HP purification Kit. New Zealand rabbits was immunized with the purified protein. The antisera titer to native recombinant protein was 1:6400 by indirected enzyme-linked immunosorbent assay (ELISA). HEp-2 cells, a kind of typical cell model, was used in the adhesion and adhesive inhibition experiments to evaluate the adhesion of the fusion protein from IMT5155. Meanwhile the fusion protein were co-cultured with HEp-2 cells to detect cytopathic effect (CPE). The purified protein did not cause CPE and death of mice by intraperitoneal injection, suggesting it is avirulent. The results demonstrated the fusion protein can inhibit the adhesion of IMT5155 strain to HEp-2 cells to some extent.4. The 2-week-old chickens were vaccinated with purified recombinant protein and the whole IMT5155 strain which were both emulsionized by ISA206, respectively. Then those vaccinated chickens were challenged with APEC IMT5155. Meanwhile the unvaccinated chickens were used as control. The chickens vaccinated by purified recombinant protein suffered 60% of mortality; the chickens vaccinated by the IMT5155 strain suffered 80%, and unvaccinated and challenged chickens suffered 90%. However, unvaccinated and unchallenged chickens were all survival. Compared with unvaccinated chickens, the two vaccinated chickens had some mild gross lesions in heart and liver. We concluded that the purified recombinant protein have the protection against the virulent E.coli IMT5155 to some extent.