【作者】 卢艳；

【导师】 曲连东； 崔玉东；

【作者基本信息】 黑龙江八一农垦大学 ， 预防兽医学， 2012， 硕士

【摘要】 鸭巴氏杆菌病(Duck pasteurellosis)是由多杀性巴氏杆菌(Pasteurella multocida, P.m)引起鸭的一种急性败血性传染病，又称为鸭出血性败血症或鸭霍乱。多杀性巴氏杆菌为革兰氏阴性菌，不但可以感染各年龄段的鸭只，而且传播途径广泛，几乎所有的家禽和野禽都是易感者。外膜蛋白H(OmpH)是多杀性巴氏杆菌主要外膜蛋白之一，各血清型间同源性较高。OmpH位于菌体表面，是一种孔道形成蛋白，属于非特异性细菌外膜脂蛋白超家族。用纯化的天然OmpH诱导的保护率与全菌的保护率相当；且OmpH和全菌都能诱导高水平的ELISA抗体。因此，以OmpH为免疫原，制备抗OmpH的特异性单克隆抗体，并在此基础上建立特异性较强的检测方法对鸭多杀性巴氏杆菌病的诊断与检测具有重要的意义。本研究以多杀性巴氏杆菌OmpH重组蛋白为免疫原，免疫6周龄BALB/c小鼠，4次免疫后，小鼠的效价在1:64000～1:512000之间。取效价最高的小鼠脾细胞与SP2/0骨髓瘤细胞，在50%PEG3350的作用下进行融合，建立抗多杀性巴氏杆菌OmpH抗体的杂交瘤细胞。应用OmpH重组蛋白间接ELISA筛选及经过3次连续克隆，获得3株能稳定分泌抗OmpH单克隆抗体的杂交瘤细胞株，分别命名为1A9、1E9和3G7。杂交瘤细胞的腹水单克隆抗体间接ELISA效价1A9和1E9是1.6×10~4，3G7是3.2×10~4；单克隆抗体亚类鉴定表明，1A9、1E9和3G7均为IgG1亚类，轻链均为κ链。经Western blot及IFA证实，3株单克隆抗体与靶抗原具有良好的反应性，且不与鸭疫里默氏杆菌、鸭大肠杆菌病的阳性血清发生反应，说明都具有良好的特异性。将1A9、1E9和3G7体外培养连续传30代，每隔5代检测效价，说明其仍能稳定分泌抗体。本研究在研制出多杀性巴氏杆菌特异性单克隆抗体的基础上以OmpH重组蛋白为包被抗原，被检血清为阻断剂，1A9、1E9和3G7单克隆抗体为一抗，羊抗鼠酶标抗体为二抗，进行阻断ELISA试验，确定以3G7单克隆抗体为最佳一抗。通过对阻断ELISA各条件的优化，最终确定了OmpH重组蛋白工作浓度为1.0μg/mL；待检血清的工作浓度为1:5；单克隆抗体的工作浓度为1:500；酶标二抗的工作浓度为1:30000建立了能检测多杀性巴氏杆菌特异性抗体的阻断ELISA方法。通过对60份鸭多杀性巴氏杆菌阴性血清样品阻断ELISA检测结果进行统计学分析，确定了该方法的判定标准：当阻断率PI≥37.6%时，血清样品判为阳性，阻断率PI <29.13%时，血清样品判为阴性。阻断率PI在两者之间时，判为可疑。应用该方法对鸭传染性浆膜炎、鸭大肠杆菌病、鸭肝炎、鸭H5N1流感、鸭网状内皮增生症、鸭呼肠孤病的阳性血清进行了检测，结果证明该方法具有良好的特异性。应用该方法对10份鸭阳性血清和10份鸭阴性血清进行检测，结果证明该方法具有良好的敏感性。批内、批间重复试验变异系数均小于10%，说明该方法具有良好的重复性。应用该方法对黑龙江省部分地区采集60份鸭血清样品进行检测，与Western blot检测结果对比，两者的符合率为91.38%。更多还原

【Abstract】 Duck pasteurellois is caused an acute septic infectious disease by Pasteurella multocida, asduck hemorrhagic septicemia or cholera. Pasteurella multocida is a Gram-negative bacteria thatinfect ducks of all ages, almost all of the poultry and wild birds are susceptible, especiallychickens, ducks and geese. The outer membrane protein H is the major outer membrane proteinsof Pasteurella multocida, high homology among the serotypes. OmpH is a pore-forming proteinrelated to the superfamily of the nonspecific bacterial porins. Purified natural OmpH induced goodprotection rate as the protection rate of whole cell lysate induced; OmpH and whole cell lysatecould induce high levels of ELISA antibody. So Pasteurella multocida OmpH recombinantprotein as immunogen, preparation of OmpH monoclonal antibodies, Using this antibody, a blockELISA method for the detection of Pasteurella multocida was developed, that is of greatsignificance for diagnosis and detection of duck pasteurellois.6-week-old female BALB/c mice were immunized by Pasteurella multocida OmpHrecombinant protein. spleen cells were fused with myeloma cell Sp2/0in50%polyethylene glycol3350after four immunizations, established hybridoma anti-Pasteurella multocida OmpH antibody.The test of indirect ELISA is used to screen hybridoma cells and limited dilution method wasperformed to subclone the positive clones. Three strains of hybridoma secreted MAb againstPasteurella multocida OmpH are obtained after three subcloning, and are designated as1A9,1E9and3G7. The titers of obtained MAbs from the mouse ascitic fluid are1.6×10~4～3.2×10~4inindirect ELISA test. By using the immunoglobulin subtypes kit, the antibodies are all IgG1with κlight chain. Western blot and IFA Confirme three Mabs are specific to Pasteurella multocidaOmpH.1A9,1E9and3G7in vitro continuous30generations, every5detection efficiency showthat they could secrete stably antibodies.A blocking ELISA based on the3G7monoclonal antibody was developed to detectantibodies to Pasteurella multocida. The best concentration of antigen that was selected byphalanx experiment was1.0μg/mL; serum dilution and monoclonal antibody dilution were1:5and1:500; the best concentration dilution of second antibody marked with HRP was1:30,000. Thethreshold value of the blocking ELISA was conformed according to the detection result of60negative sera. It was positive when PI was more than37.6%, it was negative when PI was lessthan29.13%. Positive serums to6usual pathogens and of duck were tested by blocking ELISA,the results showed that the blocking ELISA with good specificity. Intro-batch test and Inter-batchtest were less than10%, with good reproducibility.10positive serums and10negative serumswere tested by blocking ELISA, the results showed that the blocking ELISA with good sensitivity.60sera samples collected in helongjiang province were tested by blocking ELISA, the coincidencerate between of ELISA and Western blot was91.3%.更多还原