The Adherence Characterization Of Recombinant Type 1 Pili From Avian E. Coli And FimH Gene Cloning

Type 1 pili (fimbriae) are the most common adhesive appendages of E. coli, which are filamentous.It was encoded from fimH geneome,including fimB,fimE,fimA,fimI,fimC,fimD,fimF,fimG and fimH gene ,that fimA and fimH was its structurai gene.Type 1 pilus was pathogenic factor f chicken Escherichia coli.Through its adherence. of the trachea epidermis cell of respiratory road and bacteria can colonize and mobile ,thus it received invasive road .The adherence effection of the adherence characterization was related to fimH protein,and was leaded by the mannose.This research to explain the effection of fimH gene and bacteria adherence processiong of its encoded fimH protein,and analyzed the defference of fimH gene and appraised its meaning of its immunogenicity.With the two constructed recombinant bacteria of which included chicken operon gene of chicken Escherichia coli 1 pilus CZYR9(pBG-HB101, including fimB,fimE,fimA,fimI,fimC,fimD,fimF,fimG,flaw fimH) and CZYR10 (pBH-HB101, pBG-HB101, including fimB,fimE,fimA,fimI,fimC,fimD,fimF,fimG and the fimH entire gene), by electron microscope ,to observe the 1 pilus of two recombinant avian Escherichia coli strain, it was discovered that its expressed 1 pilus in the nutrition bouillon in the culture medium in 37℃, but did not express the pilus in the culture medium in 18℃. Using the adherency experiment of CZYR9 and CZYR10 to the chicken trachea mucous membrane in the culture medium in 37℃, and by scanning electron microscope observation, it was discovered that CZYR10 may stick to chicken trachea in vivo and in vitro, but which lacks this kind of adhesive effection in the culture medium in 18℃, but CZYR9 can not stick even if in 37℃; Using the adherency suppression experiment of the anti 1 pilus blood serum to determine CZYR10 to chicken trachea stage adherency specificity in vitro, the result showed the anti-1 pilus blood serum can obviously suppress the strain to the trachea epidermis adherency. Using the mannose suppression experiment,it was discovered that CZYR10 to the trachea epidermis adhesive effection may be remarkably suppressed by the mannose ,but CZYR9 regardless of which does not need mannose processing not to have the adhesive effection, which indicated that the mannose was a vital role to lead the mycelium to he trachea mucous membrane epithelial cell acceptor, FimH is related to the adherency,but cannot suppress the strain to the trachea epidermis coherence with the mannose , it indicated this mycelium the adhesive effection does not lie by the mannose leading.Extracting the template of genome from avian pathogenicEscherichia coli MG(011), GL7(O78) and YR10(O18) separately ,according to the sequence published on the Genbank, designing , synthesizing one pair of primer to amplify its fimH gene of 1 pilus and sequencing, in comparsion with overseas standard fimH gene sequence of 1 pilus of the K-12 strain.The resulted that the homology of nucleotide was 97.89%,97.56%,98.22%,respectively, and that the similarity of amino acid was 98.33%,98.33%,99.00%,respectively.From forecasting in the amino acid sequences be seen, there were only three and five sites changed, O18 to K-12 of which were the first was A in the forty-eighth site ,instead of V,the second site was P in the one hundred and ninety-sixth site,instead of R., and the third site was T in the two hundred and twenty-second site,instead of H.O78 and O18 to K-12 of which were the first site was A in the forty-eighth site,instead of V,the second site was S in the ninety-first site,instead of N,the third site was N in the ninety-ninth site,instead of S,the fourth site was P in the one hundred and ninety-sixth site,instead of R and the fifth site was T in the two hundred and twenty-second site,instead of H.And the translation of amino acid sequences wasn,t affected other changes of nucleotide sequences .It forecasted and anlysized the FimH protein level of the atlas of two structure, antigen epitope and hydrophilicity by DNASTAR, which was discovered basic similarity of theirs,which was explained the variation of the fimH gene sequence and amino acid sequence didn,t affect the structure of the FimH protein.With the bacteria strains of 1 pilus (the entire gene recombination, wild and flaw fimH gene recombination), after raising in the large capacity , three extracted and purified monovalent 1 pilus (CZYR10,YR10,CZYR9)oil-emulsified vaccines was prepared.Seventy-five one-day-old egg chickens was randomly divided into five groups (the average of fifteen each group).After seven days.the fisrt ,second and third group chickens were vaccinated with oil-emulsified vaccines 0.5ml(containg 0.25mg), respectively. The fourth and fifth group were used as positive and negative controls.Animals were boosted one week post primary immunization . After forty-eight hours, the strain (including 1 pilus ) was raised in the nutrition bouillon in 37℃.which was contented for 1×1010 (CFU/ml).Then chickens were challengeded by YR10(O18), its dosage is 0.5m(lconluding 0.5mg). After challengeding YR10(O18) two weeks later, the mortality of vaccinated groups were 26.67%, 13.33%, 33.33%, respectively and the mortality of unvaccinated groups were 73.33%, 0,respectively;.All experimental groups, in the liver, pericardium and air sacs lesion change from organism observation,vaccinated chicken group was slighter than unvaccinated chicken group,difference remarkably(P<0.05).This indicated that two kinds of recombination 1 pilus vaccines had the function of immunity protection, but the force of protection of recombination 1 pilus vaccine is lower than wild 1 pilus vaccine,moreover, between the entire gene of recombination 1 pilus protein and the flaw fimH gene of recombination 1 pilus ,the protection strength of vaccine also had no differences obviously.