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摘要：为确定产毒素性多杀性巴氏杆菌在猪群的感染情况和对猪群进行性萎缩性鼻炎发病情况开展调查，本研究以大肠杆菌表达后纯化的重组产毒素性多杀性巴氏杆菌皮肤坏死毒素PMT蛋白为包被抗原，通过条件优化建立优化了检测产毒素性多杀性巴氏杆菌感染抗体的间接ELISA方法。经试验得出，2µg/ml为最佳抗原包被浓度，37℃ 2h是最佳包被时间，37℃ 3h是最佳封闭时间，待检血清最佳稀释度为1：100，血清最佳作用时间为37℃ 1h，酶标抗体SPA最佳稀释度为1：10000，作用时间为0.5 h，底物最佳显色时间为37℃ 5min。30份PAR阴性血清间接ELISA检测结果的统计学分析显示：本方法的判定标准是当OD450≤0.2904时判为阴性，OD450≥0.3248时判为阴性，0.2904＜OD450＜0.3248时判为可疑。特异性和可重复性实验证明建立的该检测方法特异性强，可重复性好，完全可以用于临床样品的检测。28597
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毕业论文关键词：猪进行性萎缩性鼻炎；产毒素性多杀性巴氏杆菌；PMT蛋白；间接ELISA
Development and Optimization of an Indirect ELISA for Antibody Detection against Toxigenic Pasteurella Multocida
Abstract: To provide a serological method for detecting antibodies against toxigenic Pasteurella Multocida, an indirect ELISA was developed based onpurified PMT protein expressed in E. coli . The conditions for each step were optimized. 2µg/ml was the best concerntration of the coating antigen and 2 h at 37 °C was the besr time of the coating antigen; 3 h at 37 °C was the best time of blocking buffers; sera samples dilution were 100 fold and incubated 1h at 37 °C; SPA was diluted 10000 fold and incubated 0.5 h at 37 °C; the optimal reaction time of TMB was 5 min at 37 °C. Statistically analyzed the detection results of thirty toxigenic Pasteurella Multocida negative sera samples, that revealed when the samples presenting OD450≤0.2904 were considered negative; samples with a calculated OD450≥0.3248 were rated positive and those presenting between 0.2904 and 0.3248 were considered inconclusive. Specificity and repetition tests confirmed that this assay is highly specific and repeated, which can be used to diagnose clinical samples.
Key words: progressive atrophic rhinitis；toxigenic Pasteurella multocida；PMT protein；indirect ELISA
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1 材料与方法2
1.1 材料 2
1.2 抗原蛋白的制备 3
1.3 间接ELISA条件优化3
2 结果与分析4
2.1 PMT蛋白的纯化4
2.2 间接ELISA方法的建立与优化 4
2.3 间接ELISA临界值的确定7
2.4 间接ELISA的重复性试验8
2.5 间接ELISA的特异性试验8
2.6 临床血清样品的检测9
3 讨论9
致谢10
参考文献10
产毒素性多杀性巴氏杆菌感染抗体间接ELISA方法的建立与优化
多杀性巴氏杆菌(Pasteurella multocida, Pm)可以导致禽霍乱、牛羊败血症、猪肺疫等危害严重的畜禽传染病[1]。其中产毒素性多杀性巴氏杆菌(toxigenic Pasteurella multocida, T+Pm)在临床生产中可以导致进行性萎缩性鼻炎(progressive atrophic rhinitis, PAR)。该菌分泌的一种由toxA基因编码的皮肤坏死毒素（Pasteurella multocida toxin, PMT）引起猪的主要临床症状特征是：猪鼻炎，猪面部变形，典型的鼻甲骨萎缩变化，营养代谢障碍，发育迟缓，猪机体免疫系统受损导致其易感其他病原微生物，交叉感染情况严重[2-4]。该毒素分子量大小约146kDa。PAR是对现代集约化养猪场生产危害最大的传染性疾病之一，被OIE列为B类动物传染病[5]。
1830年人们第一次在德国发现PAR，现在世界各地都有其分布。PAR每年导致的经济损失惨重，可达数千万元[6]。该病可使肉猪饲料转化率降低，发育不良，增重减慢，出栏时间推迟，并使感染猪机体免疫力下降，更容易感染猪繁殖与呼吸综合征病毒、支原体、猪流感病毒和其他病原体。因此，要提高猪群生产性能就必须对该病进行有效的控制[7]。此时就必须使用实验室诊断技术来确认。现阶段有许多PAR的实验室诊断方法，整体研究方向趋于特异性强、敏感度高、操作简便、费用低廉。