The Study Of Anti-Fimbriae Of Type 1 From Avian E.Coli And The Fusion Expression Of The Pila Gene And The Immunogenic Study Of The Recombinant Fimbriae Proteins

1, The type 1 fimbriae was extracted from JM10 E. coli, and electrophoresed via 12% SDS-PAGE. Then the 18.0KDa band of fimbriae protein was cut, smashed and made into the oil antigen and vaccinated the BALB/c mice. The spleen cell of the BALB/c mice was extracted, and fused with SP2/O.A set of two hybridoma cell lines, 4D3, 1H11, secreting monoclonal antibodies specific to type pili of avian E.coli, were obtained by indirect ELISA .Their specificity was evaluated by the mannosesensitive hemagglutination blocking test, and immunoblotting. The antibodies from mouseascites had the titers of 10~4-10~5 and 10~3 in iELISA respectively and the mouse ascites had the titers of 2~5~2~6, 2~8~2~9 in the direct agglutination test. The type 1 pili of 46 avian Escherichia coli isolates were determined with these two monoclonalantibodies in the direct agglutination test. We concluded that the McAB can authenticate the pathological isolate quickly and simply, at the same time, the two McABs provided the standard fimbrial antibody for the later the antigenicity examination test.2. According to the published sequence of pilA from the Escherichia coli of human, one pair of primers was designed. The pilA gene was amplifyed from the chromosome DNA, and cloned into the T-vector, and determined, and linked with the pET-32a( + ), and transferred into E. coli BL21(DE3). After induced with IPTG, the results of SDS-PAGE demonstrated that the pilA protein was expressed by the recombinant expression plasmids. Then, the expression conditions of the recombinant bacterium pETF12/BL21 was groped from the time of expression, the concentration of IPTG and the excretion, and the suitable system of high expression ration was obtained. According to the qualities of fusion proteins, the recombinant protein of the type 1 of fimbriae from the JM10 pathological avian Escherichia coli was purified by the Hisbinding nickel-pillar. The purified recombinant protein especially reacted with the homologous McAB 4D3 and the heterogenous McAB 3E1-4 of serotype O78 and the multivalent antiserum YRO of serotype O18.The test authenticated that the recombinant proteins possess the antigenicity of the fimbriae, and occur the crossing reaction. According to these results, we concluded that among the type 1 of fimbriae, there are the common sites of antigen,and some relativities.3.The chicken were vaccinated with the purified recombinant proteins and the pilus fimbriae vaccines of JM10, respectively. After two weeks, those chicken were challenged with JM10 E. coli, respectively. Unvaccinated chickens suffered 87.5% mortality. The chickens vaccinated by recombinant fimbriae suffered 25% mortality when challenged with homologous E. coli, and The chickens vaccinated by the pilus fimbriae vaccines survived 87.5%. Compared with the unvaccinated chickens, the two vaccinated chickens had very mild gross lesion in the air sacs, liver and pericardial sacs. Compared between the chickens vaccinated by recombinant fimbriae and the chickens vaccinated by the pilus fimbriae vaccines, the former was more serious than the later, we concluded that the recombinant fimbriae possess the vaccinated protection character, and the protect effect of recombinant fimbriae was not a patch on that of the pilus fimbriae vaccines.