The Study On The Molecular Basis Of Pathogenicity And Scanning Of Immune Related Epitopes In The Swine Klebsiella Pneumoniae's Fimbriae

Klebsiella pneumoniae belongs to Gram-negative Enterobacteraceae, klebsiella, and it is divided into three subspecies: subsp.pneumoniae, subs p.azaenae and subsp. rhinoscleromatis. Klebsiella pneumoniae was isolated from the patent with pneumonia genuina by Friediande in 1893. Klebsiella pneumoniae is widespreaded in nature,and it is conditional pathogenic bacteria of human and animal intestinal tract and respiratory tract. It can cause pneumonia, septicemia, meningitis, liver abscess, urinary system phlegmasia etc. under the conditions of poor immunity and antibiotic-abused. It is the most important conditional pathogenic bacteria in human infection expect for Bacterium coli, and many domestic animals, fowls, wildlifes and aquatic animals as well.As a result of various kinds of antibiotic widely being used, more and more drug resistant strains of Klebsiella pneumoniae emerged, and most of them are multidrug resistant.At present, the Klebsiella pneumoniae is just second only Escherichia coli and aeruginosus Bacillus infection in nosocomial infection. In addition, the infections of Klebsiella pneumoniae usually break out due to not take immunoprophylaxis, climate change obviously, bad sanitation condition and so on.This disease has caused large economical losses.More and more proof indicated that the fimbriae plays an importament role in the process of infection due to bacterial adhere. The bacterial can adhere to the host's tissues and organs through adhesion. This is the frist step for bacterial invasion. The fimbriae of Klebsiella pneumoniae has been divided into two types,namely, fimbriae type I and typeⅢfimbriae. The fimB, fimE, fimA. fimI, fimC, fimD ,fimF, fimG , fimH and fimK encode type I fimbriae. The fimA encodes the petilar of the fimbriae. The fimH encodes the adhesin of the fimbriae. The type I is mannitose sensitive hemagglutination (MSHA).Most strains obtained from clinic have typeⅢfimbriae. The mykE, mykA,mykB, mykC,mykD and mykF encode typeⅢfimbriae. The mykA encodes the petilar of the fimbriae. The mykD encods the attachment proteins of the fimbriae. The typeⅢfimbriae is mannitose resist hemagglutination(MSRA).In our study, the purified Klebsiella pneumoniae fimbriae was used to immunize rabbits, then the antiserum was purified. The polyclonal antibody was used as target moledcule for scanning the random 12 peptides library in order to mimic the epitops, which can provide theoretical proof for the more effective diagnostic reagent and vaccine and the molecular basis for the pathogen adhering to the recieps.Three Gram-Negative bacillus were isolated from the lungs and livers of 9 dead pigs, and the morphology, cultural characters, biochemical characters of isolated strains were studied. From the results of biochemical tests, we found that they belonged to Klebsiella pneumoniae which had strong pathogenicity to mice. As the results of PCR for 16SrRNA, they were identified as Klebsiella pneumonias. Therefore, we established convenient molecular diagnostic method.Three strains containing typeⅢfimbriae without typeⅠby MSHA and MRHA were identified. The expression condition in vitro of different solid medium, liquid medium and culture time were optimized. In the result, the fimbriae can be fully expressed in vitro through three continuous passage culture as following procedure: inoculated in semisolid culture, picking up monoclone and cultruring in Minca liquid mediem (37℃, stationary culture for 48h). A pair of primers of typeⅢstructural gene were designed according to Genebank, and the results of PCR for structural gene showed 87.4% homology with the target fragments . Therefore, this novel method can offer high throughput molecular diagnosis for identification of typeⅢfimbriae. The optimized condition of fimbriae culturing and the method of convenient molecular diagnostic method can play an important role in further research of the mechanism and effect of fimbriae. As the results of DNAStar analysis, the reason for the diversity of typeⅢfimbriae of the isolated strains may be concluded as nucleotide mutation.The typeⅢfimbriae of Klebsiella pneumonias was extracted and purified, and the concentration of fimbriae was up to 20.5mg/mL. The polyclonal antibody F(+) PcAb)for typeⅢfimbriae was prepared through immunized rabbit by purified typeⅢfimbriae of Klebsiella pneumonias. The titers of F(+)PcAb tested by indirect ELISA is 1:64000. F(+)PcAb can finely specifically bind with typeⅢfimbriae of Klebsiella pneumonias by Western-blotting. F(+)PcAb and F(-)IgG was purified using octanoic acid-ammonium sulfate method, and the concentration of F(+)PcAb is 67.67mg/mL ,while the concentration of F(-)IgG is 103.51mg/mL.We subtract–screened F(+)PcAb/F(-)IgG by random 12 peptide library. Ten bacteriophage clones that can specifically bind with F(+)PcAb by indirect ELISA. Four bacteriophage clones that can both specifically bind with F(+)PcAb and competitive inhibition by fimbriae of Klebsiella pneumonia were obtained. The amino acid sequences are: MPSLNNTESKLG , QKERTKSTPMTA ,MSQPTTNKMLLS,SKKRFPNTSFRQ. Comparing the sequences of exogenous peptide and of Klebsiella pneumonias fimbriae by DNAStar analysis, we found that there is no obvious homology between the 12-peptides obtained and Klebsiella pneumonias fimbriae, which indicated their sequences mimic epitopes recognized by polyclonal antibody of Klebsiella pneumonias's fimbriae..