| Klebsiella pneumoniae, a member of Saccharomycetaceae, is a local species of bacterial in human and other animals'intestinal canal and respiratory passages. It is a conditional pathogen. Recently the drug resistance of Klebsiella pneumoniae has increased because of the rising resistance of Klebsiella and the extensive utilization of the antibiotic. The common penicillin has no effect on Klebsiella pneumoniae, which increased the difficulty of occupational therapy. Pilus is one of the most important pathogenic factors which related to the adherence and colonization of bacteria. By virtue of the adherence of adhesion to the parasitifer's epithelium, bacteria conglutinates to the host's organ, which is the most important condition of pathogenicity. The research of characteristic and monoclonal antibody of the fimbriae and the use of this McAb establish establish of ELISA detection methods have rapid, accurate, and efficient characteristics, and which will be benefit on not only revealing the pathogenesis mechanism but also on the prevention, the diagnosis and treatment of Klebsiella pneumoniae disease..Based on Kpn4 of porcine Klebsiella pneumoniae, this research aimed at getting Type 3 Fimbriae protein. The procedure was as follows:first, the Klebsiella pneumoniae was indentified that the expressed pilus was Type 3 Fimbriae through Mannose~Sensitive Hemagglutinin (MSHA) and mannose~resistant hemagglutination (MRHA), PCR, besides, the in vitro expression condition for Type 3 Fimbriae was improved. Secondly Type 3 Fimbriae protein was obtained by SephsdexG~100 gel filtration, hot elution method, and precipitation methods. After that 4～6 weeks female BALB/C mice was immuned by Kpn4. cell fusion was processed twice by using PEG. 3 hybridoma cells which secreted monoclonal antibody were obtained through indirect ELISA using Type 3 Fimbriae protein as primary antibody, and limiting dilution assay for 3 times. The 3 hybridoma cells were named as 3A1,5D8,4F2, and their titer, subtype, specificity, neutrality, stability and relative affinity were tested. Finally both high titer and neutrality active antibody were selected (5D8and4F2). The biometric of the McAb produced by the 3 hybridoma cells were evaluated by indirect ELISA. The titer of the supernatant from the culture were 1:3 028,1:2 038, and 1:1 747 respectively, while the titer of the ascites were 3.0×106,2.7×105, and 1.4×105 respectively. McAb subtype was evaluated by McAb subtype kit of mouse. The result showed that 3A1 was IgG type while 5D8 and 4F2 was IgM type. In order to evaluate the cellular activity and the ability to secret antibody of the 3 hybridoma cells, they were subcultured for 30 generations, and cryopreserved for 6 months in liquid nitrogen. The result showed that all 3 hybridoma cells could secret McAb to Type 3 Fimbriae protein, and the affinity was 3A1>5D8>4F2. The particularity test showed that the 3 McAb could only react with Type 3 Fimbriae protein, and the ABC~ELISA was developed.This research produced of Type 3 Fimbriae McAb and developed the method of ABC~ELISA from the separation, purification and indentification of Kpn4 of porcine Klebsiella pneumoniae, which will contribute to the study of etiology, molecular epidemiology, and the pathogenic mechanism and immune protection of Type 3 Fimbriae of porcine Klebsiella pneumonia. |
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