猪源肺炎克雷伯菌Ⅲ型菌毛单克隆抗体制备及ELISA检测方法的建立
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摘要
肺炎克雷伯菌(Klebsiella pneumoniae)是人与动物肠道和呼吸道常见的定植菌种,属于肠杆菌科,是一种条件性致病菌。菌毛是其重要的致病因子之一,与细菌的粘附定植相关,细菌可借助于菌毛尖端的粘附素(Adhesin)对宿主粘膜上皮细胞的粘附作用粘附到宿主组织器官,这是机体致病的首要条件。
本试验以临床分离的猪源肺炎克雷伯菌菌株Kpn4为研究对象,首先通过甘露糖敏感血凝与抵抗血凝试验、PCR鉴定该菌生长表达的菌毛类型为Ⅲ型菌毛,并改良了Ⅲ型菌毛的体外表达条件,后经SephsdexG—100凝胶过滤法、热洗脱法及酸盐沉淀法纯化得到Ⅲ型菌毛蛋白。然后以Kpn4为抗原免疫4～6周龄BALB/C雌性小鼠,运用PEG融合法,经两次细胞融合,以Ⅲ型菌毛蛋白为一抗,通过间接ELISA法筛选,有限稀释法克隆3次,得到3株可分泌Ⅲ型菌毛蛋白单克隆抗体的杂交瘤细胞株,分别命名为3A1、5D8、4F2,并对其效价、亚类、特异性、中和性、稳定性和相对亲和力进行了测定;结果表明制备出的三株稳定分泌肺炎克雷伯菌Kpn4 McAb的杂交瘤细胞株3A1、5D8和4F2腹水效价依次为1∶3.2×106、1∶2.8×105和1∶1.8×105,培养上清效价依次为1∶3002、1∶2119、1∶1769。3A1为IgG2b亚型,5D8和4F2为IgM亚型。三株McAb的相对亲和力为3A1>5D8>4F2;特异性测定结果表明,本试验所制备的肺炎克雷伯Ⅲ型菌毛McAb特异性反应显示与大肠埃希菌,副伤寒沙门菌,铜绿假单胞菌,产单核细胞李斯特杆菌,胸膜肺炎放线杆菌均无交叉反应性,具有较好的特异性。稳定性试验显示三株杂交瘤细胞都能稳定分泌Kpn4 McAb的能力。筛选效价高、中和活性好的1株单克隆抗体(3A1),建立双抗夹心ELISA检测方法,建立标准曲线,其标准曲线的回归方程为y =0.4333x -0.2223,最低检测限为62.5ng/mL,最适检测范围为62.5～4000 ng/mL。为该菌的快速诊断、噬菌体文库的展示和阻断菌毛粘附蛋白的作用机制及分子流行病学调查提供基础。
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.
本试验以临床分离的猪源肺炎克雷伯菌菌株Kpn4为研究对象,首先通过甘露糖敏感血凝与抵抗血凝试验、PCR鉴定该菌生长表达的菌毛类型为Ⅲ型菌毛,并改良了Ⅲ型菌毛的体外表达条件,后经SephsdexG—100凝胶过滤法、热洗脱法及酸盐沉淀法纯化得到Ⅲ型菌毛蛋白。然后以Kpn4为抗原免疫4～6周龄BALB/C雌性小鼠,运用PEG融合法,经两次细胞融合,以Ⅲ型菌毛蛋白为一抗,通过间接ELISA法筛选,有限稀释法克隆3次,得到3株可分泌Ⅲ型菌毛蛋白单克隆抗体的杂交瘤细胞株,分别命名为3A1、5D8、4F2,并对其效价、亚类、特异性、中和性、稳定性和相对亲和力进行了测定;结果表明制备出的三株稳定分泌肺炎克雷伯菌Kpn4 McAb的杂交瘤细胞株3A1、5D8和4F2腹水效价依次为1∶3.2×106、1∶2.8×105和1∶1.8×105,培养上清效价依次为1∶3002、1∶2119、1∶1769。3A1为IgG2b亚型,5D8和4F2为IgM亚型。三株McAb的相对亲和力为3A1>5D8>4F2;特异性测定结果表明,本试验所制备的肺炎克雷伯Ⅲ型菌毛McAb特异性反应显示与大肠埃希菌,副伤寒沙门菌,铜绿假单胞菌,产单核细胞李斯特杆菌,胸膜肺炎放线杆菌均无交叉反应性,具有较好的特异性。稳定性试验显示三株杂交瘤细胞都能稳定分泌Kpn4 McAb的能力。筛选效价高、中和活性好的1株单克隆抗体(3A1),建立双抗夹心ELISA检测方法,建立标准曲线,其标准曲线的回归方程为y =0.4333x -0.2223,最低检测限为62.5ng/mL,最适检测范围为62.5～4000 ng/mL。为该菌的快速诊断、噬菌体文库的展示和阻断菌毛粘附蛋白的作用机制及分子流行病学调查提供基础。
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.
引文
[1] X iong Z,Zhu D,Zhang Y,et al.Extended-spectrum beta-lactamase in K lebsiella pneumoniae and Escherichia coli isolates[J].Chin med J,2002,82(21):1476.
[2] Sahly H, Podschun R, Ullmann U. Klebsiella infections in the immunocompromised host[J].Adv Exp Med Biol. 2000;479:237-249.
[3] Hooi LN, Looi I, Ng AJ. A study on community acquired pneumonia in adults requiring hospital admission in Penang[J]. Med J Malaysia, 2001, 56(3):275-284.
[4]文细毛,任南,徐秀华,等.全国医院感染监控网医院感染病原菌分布及耐药性分析[J].中华医院感染学杂志,2002,12(4):241.
[5] Jeena P, Thompson E, Nchabeleng M, et al. Emergence of multi-drug-resistant Acinetobacter anitratus species in neonatal and paediatric intensive care units in a developing country: concern about antimicrobial policies[J]. Ann Trop Paediatr, 2001,21(3):245-251.
[6] Fadeeva GB, Sterkhova GV, Sidorenko SV, et al. Etiology and microbiological diagnosis of nosocomial pneumonia in newborns[J]. Antibiot Khimioter, 2001,46(5):17-23.
[7] Boukadida J, Salem N, Hannachi N, et al. Genotypic exploration of a hospital neonatal outbreak due to klebsiella pneumoniae producing extended- spectrumbetalactamase[J].Arch Pediatr, 2002,9(5):463-468.
[8] Tsai MH, Lu CH, Huang CR, et al. Bacterial meningitis in young adults in southern taiwan: clinical characteristics and therapeutic outcomes[J].Infection. 2006,34(1):2-8.
[9] Kim SH, Wei CI, Tzou YM,et al. Multidrug-resistant Klebsiella pneumoniae isolated from farm environments and retail products in Oklahoma[J]. J Food Prot. 2005,68(10): 2022-2029.
[10]彭广熊,熊焰.大熊猫肺炎克雷伯氏菌的研究现状[J].四川畜牧兽医,2000, 27(113):86-87.
[11]陈永林,关孚时,朱飞兵,等.引起大熊猫腹泻的克雷伯氏菌强毒株[J].中国兽医杂志,1997,31(2):34-36.
[12] Buxton A,Fraser G. Animal Microbiology[M].Blackwell Scientific Publications LTD.1977:119.
[13] J.H吉莱斯皮等著,胡祥璧等译.家畜传染病[M](第七版).北京:农业出版社,1988:70-71.
[14]杨留战,等.哺乳仔猪克雷伯氏菌肺炎杆菌的调查[J].中国兽医科技,1989, (1):17-18.
[15]朱煜兰.鸭肺炎克雷伯氏菌病的诊疗[J].中国兽医科技,1989,(12):27-28.
[16]曹荔能.麝鼠克雷伯氏菌病的诊治[J].中国兽医科技,1989,(4):27-28.
[17]彭远义,等.鸡肺炎克雷伯氏菌的分离鉴定[J].西南农业大学学报,1995, 17(2):166-168.
[18]陈永林,张成林,胥哲.大熊猫克雷伯氏菌病的诊断[J].中国兽医杂志, 2002,38(8):48.
[19]徐海圣,舒妙安.中华鳖肺炎克雷伯氏菌病的病原研究[J].浙江大学学报(理学版),2002,29(6):702-706.
[20]陶锦华,李康然,韦平.石龟肺炎克雷伯氏菌感染的诊断与防治[J].广西畜牧兽医,2002,18(6):20-21.
[21]郭定宗,胡薛英,杜有顺,等.金丝猴肺炎克雷伯氏菌及埃希氏大肠杆菌败血症的病理学观察[J].华中农业大学学报,2001,20(1):60-62.
[22]肖家美,闫喜军,赵艳.乌苏里貉克雷伯氏菌病的诊断[J].特产研究,2003, (1):30-31.
[23]李桂平,衡江鸿.獭兔抗药性肺炎克雷伯氏菌病的防治[J].中国养兔杂志,1998,(2):5-6.
[24]张海艇.小鼠由肺炎克雷伯氏菌引发疫病的报告[J].黑龙江畜牧兽医,1998,(6):32.
[25] Ronald A The etiology of urinary t ract infection:t raditionaland emerging pathogens[J].Dis Mon,2003,49 (2):71-82.
[26] Craven DE.What is healthcare 2 associated pneumonia,andhow should it be t reated [J].Curr Opin Infect Dis,2006,19(2):153-160.
[27] Yokochi T,Nakashima I,Nagase F,et al.Further studiesof the polysaccharide ofKlebsiella pneumoniae possessingst rong adjuvanticity.III.Augmentation of the antibody response to subcutaneously injected sheep red blood cells by the adjuvant polysaccharide[J].Microbiol Immunol,1982,26(9):843-852.
[28] Kabha K,Schmegner J,Keisari Y,et al.SP-A enhancesphagocytosis of Klebsiella by interaction with capsular polysaccharides and alveolar macrophages[J].Am J Physiol,1997,272(2 Pt 1):L344-L352.
[29] de Lorenzo V,Martinez JL Aerobactin production as a viru2 lence factor: a reevaluation [J].Eur J Clin Microbiol Infect Dis,1988,7 (5):621-629.
[30] Nassif X, Sansonetti PJ.Correlation of the virulence of Kleb2siella pneumoniae K1 and K2 with the presence of a plasmidencoding aerobactin[J].Infect Immun,1986,54 (3):603-608.
[31] McCallum, KL, Schoenhals G, Laakso D, et al. A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing[J]. Infect Immun ,1989,57(12):3816-3822.
[32] Merino S, Camprubi S, Alberti S, et al. Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing[J]. Infect Immun, 1992, 60(6):2529- 2535.
[33] Podschun R, Ullmann U. Klebsiella spp as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev,1998, 11(4):589-603.
[34] Yokochi T,Nakashima I,Nagase F,et al.Further studiesof the polysaccharide of Klebsiella pneumoniae possessingst rong adjuvanticity.III.Augmentation of the antibody response to subcutaneously injected sheep red blood cells by the adjuvant polysaccharide[J].Microbiol Immunol,1982,26(9):843-852.
[35] Yoshida K,Mat sumoto T,Tateda K,et al.Protection against pulmonary infection with Klebsiella pneumoniae in mice by interferon2gamma through activation of phagocytic cells and stimulation of production of other cytokines[J].J Med Microbiol,2001,50(11):959-964.
[36] de Lorenzo V, Martinez JL. Aerobactin production as a viru2 lence factor:areevaluation [J].Eur J Clin Microbiol Infect Dis,1988, 7 (5):621-629.
[37] Nassif X,Sansonetti PJ.Correlation of the virulence of Kleb2 siella pneumoniae K1 and K2 with the presence of a plasmid encoding aerobactin[J].Infect Immun, 1986,54(3):603-608.
[38] Hooi S H,Hooi S T.Culture2proven bacterial keratitis in a Malaysian general hospital [J].Med J Malaysia,2005,60(50):6142623.
[39] Miguel Regue ,Beat riz Hita,Nuria Pique,et al.A Gene,uge,Is Essential for Klebsiella pneumoniae Virulence[J].Infectionand immunity,2004,1:54261.
[40] Wit kowska D,Mieszala M,Gamian A,et al.Major st ruct uralproteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed f -rom t heir immunochemical characterization[J].FEMS Immu2 nol Med Microbiol,2005,45 (2):221230.
[41]彭广能,熊焰.大熊猫肺炎克雷伯氏菌的研究现状[J].四川畜牧兽医,2000,27 (113):86287.
[42] J agnow J,Clegg S. Klebsiella pneumoniae mrkD2mediated bio2 film formation on ext racellular mat rix2and collagen2coated sur2 faces[J].Microbiology, 2003, 149 (Pt9):239722405.
[43] Tricia A,Douglas B,Hornick.The Type 3 Fimbrial Adhesin Gene (mrkD) of Klebsiella Species Is Not Conserved among All Fimbriate St rains[J].Infection and immunity,1994,10 (62) :418624191.
[44] Bergogne Berezin E Treatment and prevention of nosocomial pneumon[J]. Chest,1995,108:26234.
[45] Langstraat J, Bohse M, Clegg S Type 3 fimbrial shaft (MrkA) ofKlebsiella pneum oniae, but not the fimbrial adhesin (MrkD),facili2 tates biofilm formation I nfect I mmun,2001,69(9) 58052 5812.
[46] Jagnow J,Clegg S.Klebsiella pneumoniae MrkD2 mediated biofilm formati on on extracellular matrix2 and collagen2 coated surfaces Mi2 cr obi ol ogy,2003,149 (Pt9).23972 2405.
[47] Langst raat J,Bohse M,Clegg S Type 3 fimbrial shaf t (mrkA) of Klebsiella pneumoniae,but not the fimbrial adhesin (MrkD),facilitates biofilm formation[J].Infect Immun,2001,69 (9):580525812.
[48] Ann2mari T,Ritva V,Steven C Binding of t he Type 3 Fimbri2 ae of Klebsiella pneumoniae to Human Endot helial and Urinary Bladder Cells [J].Infection and immunity,1997,4(65):15462 1549.
[49] Gerlach G F,Allen B L,Clegg S. Type 3 fimbriae among en2 terobacteria and t he ability of spermidine to inhibit MR/ K he2 magglutination[J ].Infect Immun, 1989,57:2192224.
[50] Darfeuille2Michaud A,J allat C,Aubel D,et al R2plasmid2en2 coded adhesive factor in Kl ebsiella pneumoniae st rains respon2 sible for human nosocomial infections[J].Infect Immun,1992,60:44255.
[51] Di Martino P,Livrelli V,Sirot D,et al.A new fimbrial antigen harbored by CAZ 25/ SHV242producing Klebsiella pneumoniaest rains involved in nosocomial infections[J].Infect Immun,1996,64:226622273.
[52]王自良,王建华,杨艳艳,等.抗氯霉素单克隆抗体杂交瘤细胞株的筛选及金标试纸检测方法的建立[J].西北农林科技大学学报(自然科学版),2006,34(5):6-11.
[53]汪招雄,何启盖,刘丽娜,等.检测伪狂犬病病毒双抗体夹心间接ELISA方法的建立与应用[J].中国预防兽医学报,2006,28(2):220-225.
[54]祝令伟,冯书章,刘军,等.肠出血性大肠杆菌O157:H7主要保护性抗原单克隆抗体的研制及特性分析[J].中国兽医学报,2006,26(3):275-277.
[55]胡薛英,谷长勤,程国富,等.应用单克隆抗体的免疫组织化学法研究雏鸭体内鸭瘟病毒的分布[J].中国预防兽医学报,2006,28(3):320-322.
[56]徐耀辉,焦新安,胡青海,等.酶标单抗阻断ELISA检测鸡白痢和鸡伤寒抗体[J].中国兽医学报,2006,26(2):140-143.
[57] Beate G R,Sandra A B,Carol K H,et al Cef tiofur Sodium Mon2oclonal antibody development and cross2reactivity studies wit hst ructurally related cephalosporins[J]. J Agric Food Chem,1996,44:622-627.
[58] David L B,Ronald GB,Anne H B,et al.Monoclonal antibody formulti2residue ELISA of benzimidazole ant helmintics in liver [J].Journal of Agricultural Food Chemist ry,1994,42:1588-1594.
[59] Dixon H D E,Katz S E. Competitive direct enzyme2linked immu2 nosorbent screening assay for t he detection of sulfamet hamine contamination of animal feeds[J].J Assoc Off Anal Chem,1991,74 (5):784-789.
[60] Elliot t C T,Baxter A,Haasnoot W Development of a dual label time2resoled fluoroimmunoassay for t he detection of bagonist s in cat tle urine[J].Food & Agricultural Immunology,1996,8:219-227.
[61] Starick E ,Werner O , Schirrmeier H,et al.Establishment of A Competitive EL ISA (cEL ISA) System for t he Detection of Influ2 enza A Virus Nucleoprotein Antibodies and it s Application to Field Sera f rom Different Species[J].J Vet Med B Infect Dis Vet Public Healt h ,2006,53 (8):370-375.
[62] Stone D M,Norton L K,Davis W C. Modulation of bovine Leu2 kemia virus2associated spontaneous lymphocyte proliferation by monoclonal antibodies to lymphocyte surface motecules [J].ClinImmunol Immunopat hol,1997,83(2): 156-164.
[63] Zhang A,Jin M,Liu F,et al.Development and evaluation of aDAS2ELISA for rapid detection of avian influenza viruses[J].A2 vian Dis,2006,50(3):325-330.
[64] PATRICK DI MARTINO,VALERIE LIVRELLI,DANIELLE SIROT,et al.A New Fimbrial Antigen Harbored by CAZ-5/SHV-4-Producing Klebsiella pneumoniae Strains Involved in Nosocomial Infections[J].Infection and immunity,1996,64(6): 2266-2273.
[65]沈关心,周汝麟.现在免疫学实验技术(第二版)[M].湖北:湖北科学技术出版社,2002.
[66]朱立平,陈血清.免疫学常用实验方法(第一版)[M].北京:人民军医出版社,2000.
[67]房海著.大肠埃希氏菌[M].河北:河北科学技术出版社,1997.
[68] Gerlach G F,Allen B L,Clegg S.Type 3 fimbriae among enterobacteria and the ability of spermidine to inhibit MR/K hemagglutination[J].Infect Immun,1989, 57:219-224.
[69] Ann-mari T,Ritva V,Steven C.Binding of the Type 3 Fimbriae of Klebsiella pneumoniae to Human Endothelial and Urinary Bladder Cells[J].Infection andimmunity,1997,4(65):1546–1549.
[70] Darfeuille-Michaud A,Jallat C,Aubel D,et al.R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible for human nosocomial infections[J].Infect Immun,1992,60:44-55.
[71] BRETK,PURCELL,JANET PRUCKLER,STEVEN CLEGG.Nucleotide Sequences of the Genes Encoding Type 1 Fimbrial Subunits of Klebsiella pneumoniae and Salmonella typhimurium[J].JOURNAL OF BACTERIOLOGY, 1987,169(12):5831-5834.
[72] BRADLEY L.ALLEN,GERALD-F.GERLACH,STEVEN CLEGG.Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae[J].Bacteriology,1991,173(12):916-920.
[73] PATRICK DI MARTINO,VALERIE LIVRELLI,DANIELLE SIROT,et al.A New Fimbrial Antigen Harbored by CAZ-5/SHV-4-Producing Klebsiella pneumoniae StrainsInvolved.inNosocomialInfections[J].Infectionandimmunity,1996,64(62):2266-2273.
[74]周宗安,等译.单克隆抗体技术手册[M].南京:南京大学出版社,1991:41-48.
[75]王世若,王兴龙,韩文瑜.现代动物免疫学(第二版)[M].长春:吉林科学技术出版社,2001:466-468.
[76] Fischer CA:Retinal and retinochoroidal lesions in early neuropathic canine distemper,J.Am.Vet,Med,Assoc.1971,158:740-752.
[77] Hunt RD,Ferrell JF,Thompson SW,et al:A histochemical comparison of the inclusion bodies of can distemper and infections canine Hepatitis.Am.J. Vet.Res. 1963, 24:1284-1255.
[2] Sahly H, Podschun R, Ullmann U. Klebsiella infections in the immunocompromised host[J].Adv Exp Med Biol. 2000;479:237-249.
[3] Hooi LN, Looi I, Ng AJ. A study on community acquired pneumonia in adults requiring hospital admission in Penang[J]. Med J Malaysia, 2001, 56(3):275-284.
[4]文细毛,任南,徐秀华,等.全国医院感染监控网医院感染病原菌分布及耐药性分析[J].中华医院感染学杂志,2002,12(4):241.
[5] Jeena P, Thompson E, Nchabeleng M, et al. Emergence of multi-drug-resistant Acinetobacter anitratus species in neonatal and paediatric intensive care units in a developing country: concern about antimicrobial policies[J]. Ann Trop Paediatr, 2001,21(3):245-251.
[6] Fadeeva GB, Sterkhova GV, Sidorenko SV, et al. Etiology and microbiological diagnosis of nosocomial pneumonia in newborns[J]. Antibiot Khimioter, 2001,46(5):17-23.
[7] Boukadida J, Salem N, Hannachi N, et al. Genotypic exploration of a hospital neonatal outbreak due to klebsiella pneumoniae producing extended- spectrumbetalactamase[J].Arch Pediatr, 2002,9(5):463-468.
[8] Tsai MH, Lu CH, Huang CR, et al. Bacterial meningitis in young adults in southern taiwan: clinical characteristics and therapeutic outcomes[J].Infection. 2006,34(1):2-8.
[9] Kim SH, Wei CI, Tzou YM,et al. Multidrug-resistant Klebsiella pneumoniae isolated from farm environments and retail products in Oklahoma[J]. J Food Prot. 2005,68(10): 2022-2029.
[10]彭广熊,熊焰.大熊猫肺炎克雷伯氏菌的研究现状[J].四川畜牧兽医,2000, 27(113):86-87.
[11]陈永林,关孚时,朱飞兵,等.引起大熊猫腹泻的克雷伯氏菌强毒株[J].中国兽医杂志,1997,31(2):34-36.
[12] Buxton A,Fraser G. Animal Microbiology[M].Blackwell Scientific Publications LTD.1977:119.
[13] J.H吉莱斯皮等著,胡祥璧等译.家畜传染病[M](第七版).北京:农业出版社,1988:70-71.
[14]杨留战,等.哺乳仔猪克雷伯氏菌肺炎杆菌的调查[J].中国兽医科技,1989, (1):17-18.
[15]朱煜兰.鸭肺炎克雷伯氏菌病的诊疗[J].中国兽医科技,1989,(12):27-28.
[16]曹荔能.麝鼠克雷伯氏菌病的诊治[J].中国兽医科技,1989,(4):27-28.
[17]彭远义,等.鸡肺炎克雷伯氏菌的分离鉴定[J].西南农业大学学报,1995, 17(2):166-168.
[18]陈永林,张成林,胥哲.大熊猫克雷伯氏菌病的诊断[J].中国兽医杂志, 2002,38(8):48.
[19]徐海圣,舒妙安.中华鳖肺炎克雷伯氏菌病的病原研究[J].浙江大学学报(理学版),2002,29(6):702-706.
[20]陶锦华,李康然,韦平.石龟肺炎克雷伯氏菌感染的诊断与防治[J].广西畜牧兽医,2002,18(6):20-21.
[21]郭定宗,胡薛英,杜有顺,等.金丝猴肺炎克雷伯氏菌及埃希氏大肠杆菌败血症的病理学观察[J].华中农业大学学报,2001,20(1):60-62.
[22]肖家美,闫喜军,赵艳.乌苏里貉克雷伯氏菌病的诊断[J].特产研究,2003, (1):30-31.
[23]李桂平,衡江鸿.獭兔抗药性肺炎克雷伯氏菌病的防治[J].中国养兔杂志,1998,(2):5-6.
[24]张海艇.小鼠由肺炎克雷伯氏菌引发疫病的报告[J].黑龙江畜牧兽医,1998,(6):32.
[25] Ronald A The etiology of urinary t ract infection:t raditionaland emerging pathogens[J].Dis Mon,2003,49 (2):71-82.
[26] Craven DE.What is healthcare 2 associated pneumonia,andhow should it be t reated [J].Curr Opin Infect Dis,2006,19(2):153-160.
[27] Yokochi T,Nakashima I,Nagase F,et al.Further studiesof the polysaccharide ofKlebsiella pneumoniae possessingst rong adjuvanticity.III.Augmentation of the antibody response to subcutaneously injected sheep red blood cells by the adjuvant polysaccharide[J].Microbiol Immunol,1982,26(9):843-852.
[28] Kabha K,Schmegner J,Keisari Y,et al.SP-A enhancesphagocytosis of Klebsiella by interaction with capsular polysaccharides and alveolar macrophages[J].Am J Physiol,1997,272(2 Pt 1):L344-L352.
[29] de Lorenzo V,Martinez JL Aerobactin production as a viru2 lence factor: a reevaluation [J].Eur J Clin Microbiol Infect Dis,1988,7 (5):621-629.
[30] Nassif X, Sansonetti PJ.Correlation of the virulence of Kleb2siella pneumoniae K1 and K2 with the presence of a plasmidencoding aerobactin[J].Infect Immun,1986,54 (3):603-608.
[31] McCallum, KL, Schoenhals G, Laakso D, et al. A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing[J]. Infect Immun ,1989,57(12):3816-3822.
[32] Merino S, Camprubi S, Alberti S, et al. Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing[J]. Infect Immun, 1992, 60(6):2529- 2535.
[33] Podschun R, Ullmann U. Klebsiella spp as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev,1998, 11(4):589-603.
[34] Yokochi T,Nakashima I,Nagase F,et al.Further studiesof the polysaccharide of Klebsiella pneumoniae possessingst rong adjuvanticity.III.Augmentation of the antibody response to subcutaneously injected sheep red blood cells by the adjuvant polysaccharide[J].Microbiol Immunol,1982,26(9):843-852.
[35] Yoshida K,Mat sumoto T,Tateda K,et al.Protection against pulmonary infection with Klebsiella pneumoniae in mice by interferon2gamma through activation of phagocytic cells and stimulation of production of other cytokines[J].J Med Microbiol,2001,50(11):959-964.
[36] de Lorenzo V, Martinez JL. Aerobactin production as a viru2 lence factor:areevaluation [J].Eur J Clin Microbiol Infect Dis,1988, 7 (5):621-629.
[37] Nassif X,Sansonetti PJ.Correlation of the virulence of Kleb2 siella pneumoniae K1 and K2 with the presence of a plasmid encoding aerobactin[J].Infect Immun, 1986,54(3):603-608.
[38] Hooi S H,Hooi S T.Culture2proven bacterial keratitis in a Malaysian general hospital [J].Med J Malaysia,2005,60(50):6142623.
[39] Miguel Regue ,Beat riz Hita,Nuria Pique,et al.A Gene,uge,Is Essential for Klebsiella pneumoniae Virulence[J].Infectionand immunity,2004,1:54261.
[40] Wit kowska D,Mieszala M,Gamian A,et al.Major st ruct uralproteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed f -rom t heir immunochemical characterization[J].FEMS Immu2 nol Med Microbiol,2005,45 (2):221230.
[41]彭广能,熊焰.大熊猫肺炎克雷伯氏菌的研究现状[J].四川畜牧兽医,2000,27 (113):86287.
[42] J agnow J,Clegg S. Klebsiella pneumoniae mrkD2mediated bio2 film formation on ext racellular mat rix2and collagen2coated sur2 faces[J].Microbiology, 2003, 149 (Pt9):239722405.
[43] Tricia A,Douglas B,Hornick.The Type 3 Fimbrial Adhesin Gene (mrkD) of Klebsiella Species Is Not Conserved among All Fimbriate St rains[J].Infection and immunity,1994,10 (62) :418624191.
[44] Bergogne Berezin E Treatment and prevention of nosocomial pneumon[J]. Chest,1995,108:26234.
[45] Langstraat J, Bohse M, Clegg S Type 3 fimbrial shaft (MrkA) ofKlebsiella pneum oniae, but not the fimbrial adhesin (MrkD),facili2 tates biofilm formation I nfect I mmun,2001,69(9) 58052 5812.
[46] Jagnow J,Clegg S.Klebsiella pneumoniae MrkD2 mediated biofilm formati on on extracellular matrix2 and collagen2 coated surfaces Mi2 cr obi ol ogy,2003,149 (Pt9).23972 2405.
[47] Langst raat J,Bohse M,Clegg S Type 3 fimbrial shaf t (mrkA) of Klebsiella pneumoniae,but not the fimbrial adhesin (MrkD),facilitates biofilm formation[J].Infect Immun,2001,69 (9):580525812.
[48] Ann2mari T,Ritva V,Steven C Binding of t he Type 3 Fimbri2 ae of Klebsiella pneumoniae to Human Endot helial and Urinary Bladder Cells [J].Infection and immunity,1997,4(65):15462 1549.
[49] Gerlach G F,Allen B L,Clegg S. Type 3 fimbriae among en2 terobacteria and t he ability of spermidine to inhibit MR/ K he2 magglutination[J ].Infect Immun, 1989,57:2192224.
[50] Darfeuille2Michaud A,J allat C,Aubel D,et al R2plasmid2en2 coded adhesive factor in Kl ebsiella pneumoniae st rains respon2 sible for human nosocomial infections[J].Infect Immun,1992,60:44255.
[51] Di Martino P,Livrelli V,Sirot D,et al.A new fimbrial antigen harbored by CAZ 25/ SHV242producing Klebsiella pneumoniaest rains involved in nosocomial infections[J].Infect Immun,1996,64:226622273.
[52]王自良,王建华,杨艳艳,等.抗氯霉素单克隆抗体杂交瘤细胞株的筛选及金标试纸检测方法的建立[J].西北农林科技大学学报(自然科学版),2006,34(5):6-11.
[53]汪招雄,何启盖,刘丽娜,等.检测伪狂犬病病毒双抗体夹心间接ELISA方法的建立与应用[J].中国预防兽医学报,2006,28(2):220-225.
[54]祝令伟,冯书章,刘军,等.肠出血性大肠杆菌O157:H7主要保护性抗原单克隆抗体的研制及特性分析[J].中国兽医学报,2006,26(3):275-277.
[55]胡薛英,谷长勤,程国富,等.应用单克隆抗体的免疫组织化学法研究雏鸭体内鸭瘟病毒的分布[J].中国预防兽医学报,2006,28(3):320-322.
[56]徐耀辉,焦新安,胡青海,等.酶标单抗阻断ELISA检测鸡白痢和鸡伤寒抗体[J].中国兽医学报,2006,26(2):140-143.
[57] Beate G R,Sandra A B,Carol K H,et al Cef tiofur Sodium Mon2oclonal antibody development and cross2reactivity studies wit hst ructurally related cephalosporins[J]. J Agric Food Chem,1996,44:622-627.
[58] David L B,Ronald GB,Anne H B,et al.Monoclonal antibody formulti2residue ELISA of benzimidazole ant helmintics in liver [J].Journal of Agricultural Food Chemist ry,1994,42:1588-1594.
[59] Dixon H D E,Katz S E. Competitive direct enzyme2linked immu2 nosorbent screening assay for t he detection of sulfamet hamine contamination of animal feeds[J].J Assoc Off Anal Chem,1991,74 (5):784-789.
[60] Elliot t C T,Baxter A,Haasnoot W Development of a dual label time2resoled fluoroimmunoassay for t he detection of bagonist s in cat tle urine[J].Food & Agricultural Immunology,1996,8:219-227.
[61] Starick E ,Werner O , Schirrmeier H,et al.Establishment of A Competitive EL ISA (cEL ISA) System for t he Detection of Influ2 enza A Virus Nucleoprotein Antibodies and it s Application to Field Sera f rom Different Species[J].J Vet Med B Infect Dis Vet Public Healt h ,2006,53 (8):370-375.
[62] Stone D M,Norton L K,Davis W C. Modulation of bovine Leu2 kemia virus2associated spontaneous lymphocyte proliferation by monoclonal antibodies to lymphocyte surface motecules [J].ClinImmunol Immunopat hol,1997,83(2): 156-164.
[63] Zhang A,Jin M,Liu F,et al.Development and evaluation of aDAS2ELISA for rapid detection of avian influenza viruses[J].A2 vian Dis,2006,50(3):325-330.
[64] PATRICK DI MARTINO,VALERIE LIVRELLI,DANIELLE SIROT,et al.A New Fimbrial Antigen Harbored by CAZ-5/SHV-4-Producing Klebsiella pneumoniae Strains Involved in Nosocomial Infections[J].Infection and immunity,1996,64(6): 2266-2273.
[65]沈关心,周汝麟.现在免疫学实验技术(第二版)[M].湖北:湖北科学技术出版社,2002.
[66]朱立平,陈血清.免疫学常用实验方法(第一版)[M].北京:人民军医出版社,2000.
[67]房海著.大肠埃希氏菌[M].河北:河北科学技术出版社,1997.
[68] Gerlach G F,Allen B L,Clegg S.Type 3 fimbriae among enterobacteria and the ability of spermidine to inhibit MR/K hemagglutination[J].Infect Immun,1989, 57:219-224.
[69] Ann-mari T,Ritva V,Steven C.Binding of the Type 3 Fimbriae of Klebsiella pneumoniae to Human Endothelial and Urinary Bladder Cells[J].Infection andimmunity,1997,4(65):1546–1549.
[70] Darfeuille-Michaud A,Jallat C,Aubel D,et al.R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible for human nosocomial infections[J].Infect Immun,1992,60:44-55.
[71] BRETK,PURCELL,JANET PRUCKLER,STEVEN CLEGG.Nucleotide Sequences of the Genes Encoding Type 1 Fimbrial Subunits of Klebsiella pneumoniae and Salmonella typhimurium[J].JOURNAL OF BACTERIOLOGY, 1987,169(12):5831-5834.
[72] BRADLEY L.ALLEN,GERALD-F.GERLACH,STEVEN CLEGG.Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae[J].Bacteriology,1991,173(12):916-920.
[73] PATRICK DI MARTINO,VALERIE LIVRELLI,DANIELLE SIROT,et al.A New Fimbrial Antigen Harbored by CAZ-5/SHV-4-Producing Klebsiella pneumoniae StrainsInvolved.inNosocomialInfections[J].Infectionandimmunity,1996,64(62):2266-2273.
[74]周宗安,等译.单克隆抗体技术手册[M].南京:南京大学出版社,1991:41-48.
[75]王世若,王兴龙,韩文瑜.现代动物免疫学(第二版)[M].长春:吉林科学技术出版社,2001:466-468.
[76] Fischer CA:Retinal and retinochoroidal lesions in early neuropathic canine distemper,J.Am.Vet,Med,Assoc.1971,158:740-752.
[77] Hunt RD,Ferrell JF,Thompson SW,et al:A histochemical comparison of the inclusion bodies of can distemper and infections canine Hepatitis.Am.J. Vet.Res. 1963, 24:1284-1255.