| Klebsiella pneumoniae is ether a kind of etiological agent of Amphixenosis or a kind of conditioned pathogen which can can cause a variety of animal disease.For a long time, the people have thinked that it was only a conditional bacteria which had not been attracted attention. But in recent years, with antibiotic has been used excessive, the majority of clinical strains isolated have multiple drug resistance.Commonly used penicillin drug of chiefly choice is ineffective for it and which have resulted in very difficulty for clinical treatment. Now, among the infection bacterial spectrum of human.Klebsiella pneumoniae has become important iatrogenic infection pathogens second only to E. coli and Pseudomonas aeruginosa. Moreover, the hazard which Klebsiella pneumoniae harm to animal is very serious. Klebsiella pneumoniae have cell antigen, k-antigen, fimbrial antigen and so on multiple antigen components. The people have considered generally that The fimbriae is one of important pathogenic factors. Klebsiella pneumoniae has two kinds of fimbriae, namely Type1 and 3 fimbriae, the both were encoded by the related gene and nearly all composed of the protein which have the characteristics of virulence factor and immunogen,so thus is always one of research objects on which domestic and foreign scholars. The research of characteristic and monoclonal antibody of the fimbriae have the vital significance on not only revealing the pathogenesis mechanism but also on the prevention, the diagnosis and treatment of Klebsiella pneumoniae disease. In this paper,The type of fimbriae of 5 avian K.pneumoniae isolates had been identificated preliminary by the way of MSHA/MRHA which according to D-mannose sensitive hemagglutinin characteristics of type 1 fimbriae and D-mannose resistance hemagglutinin characteristics type 3 fimbriae.The results showed strains Kpn6, Kpn7, Kpn8, Kpn9 possessed type 3 fimbriae, but the type of fimbriae of Kpn5 had not been able to be identificated.Then, according to type 1 and 3 fimbriae main structure sub-unit (fimA/mrkA) sequence,after two pairs of primers were future designed and synthesized, structural gene of the fimbriae of 5 srains was cloned by PCR. The results showed all of 5 avian K.pneumoniae isolates possessed type 3 fimbriae.Meanwhile the PCR detection method of type 3 fimbriae of Klebsiella pneumoniae had been established.the examination sensitivity of bacterium gene group DNA was 16.8fg/μL, while the examination sensitivity of bacterium culture was 3.7×102CFU/mL.The specificity response showed that it had no response with the reference strain.Because of which the fimbriae expression in vitro subject to the restrictions of culture conditions is very significant, therefore, the partial factors of impacting expression of type 3 fimbriae were optimized.The strain was cultivated in different mediums, temperatures, time of cultivation, pH values and methods of cultivation. The hemagglutination titer of culture were detected by the way of MRHA and culture were observated with electron microscopy,which could revealed determined the conditions expression in vitro of type 3 fimbraie Klebsiella pneumoniae. The results showed that type 3 fimbraie would gotten good growth in condition of which the strains were cultivated in improvements Minka liquid medium (pH 6.8~7.2),37℃,after 3 passage. the degree of fimbriae expression can achieve more than 90%.The hemagglutination tite of Kpn7 were the highest among 5 avian K.pneumoniae isolatesAfter fimbriae culture condition was determinated, taking strain Kpn7 as the research material, the type 3 fimbriae antigen was obtained by using the hot-bath method, and purified by the ammonium sulfate precipitation and the sucrose gradient densitied overspeed centrifugation;The rabbit was immunitied cell antigen by whole-cell antigen to prepare antiserum of Klebsiella pneumoniae. the result of the indirect ELISA showed that serum titter reached 1: 32 000,and Western blot showed a specific band. The antiserum was purified by the way of bitter-ammonium sulfate precipitation, and protein density was 3.56mg/mL by the ultraviolet spectrophotometer examination .Finally, after taking entire bacterium to immune BALB/c mouse, the result of indirect ELISA showed that serum titter reached 1: 16 000.after the last immunity, taking the spleen and the SP2/0 myeloma cell to fusion, and the fusion rate was 73.61%; Taking type 3 fimbriae protein purificated as coated antigen, screened for masculine hybrid lump cells by the indirect ELISA.The result showed the positive rate was 21.69%; To the strong and grow queckly hole to use limitedly culture method, after three clones, obtained a hybridoma cell named 3E10 which could secrete the monoclonal antibody agaist type 3 fimbriae of Klebsiella pneumoniae, The result of chromosome analysis demonstrated its amout was 97. hybridoma cell culture's and the ascites titter was 1:4 000 and 1:256 000 by the indirect ELISA.By the immunity globulin class and the subgroup appraisal, it was comfirmed to be IgG1.Prepared the monoclonal antibody ascite by using animal in vivo production, and the ascite was purified by the ammonium sulfate precipitation after using the silicon dioxide adsorption.The result of SDS-PAGE showed that purified monoclonal antibody had two belts: one was IgG heave strain, approximately 50.0kDa, and the other was the light strain, approximately 25.0kDa. Monoclonal antibodies molecular weight is about 150.0kDa. Purified ascite IgG density is 3.74mg/mL by the ultraviolet spectrophotometer,and the antibody effective density was 3.27mg/mL by Sandwich ELISA. The monoclonal antibody had a higher affinity by the indirect ELISA, and the relative affinity was 0.3μg /mL; The monoclonal antibody had no crossing reaction with the Escherichia coli, Salmonella paratyphi, Pseudomonas aeruginosa, L.monocytogenes,Actinobacillus Pleuropneumoniae. This research provided the material and layed the foundation for this bacterium etiology, the molecular epidemiologyas as well as the research of type 3 fimbriae.
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