肺炎克雷伯杆菌和嗜肺巴氏杆菌特异性抗原的筛选与鉴定

发布时间：2018-08-16 13:54

【摘要】： 肺炎克雷伯杆菌(Klebsiella pneumonia),嗜肺巴氏杆菌(Pasteurela pneumotropica)是实验动物特别是大小鼠的常见病原菌；实验动物感染后将对实验结果造成影响。我国实验动物国家标准中规定：无特定病原体(SPF)动物必须无此类细菌感染。当今对于这两种细菌的检测主要是分离培养法,该方法因灵敏度低有待改进。基于特异性抗原的血清学检测方法拥有灵敏度高,特异性强,成本低,速度快,感染后恢复期的动物也能检出等特点,是实验动物病原感染检测优选方法。我们在对肺炎克雷伯杆菌特异性抗原的研究中,采用甲醛灭活的10种小鼠常见病原菌菌体免疫小鼠制备多克隆抗体,使用双向电泳分离肺炎克雷伯杆菌的总蛋白,并以免疫印迹法(western blotting)与肺炎克雷伯杆菌多克隆抗体反应,筛选出免疫原性强的蛋白抗原；再将细菌总蛋白与其他多克隆抗体反应来排除交叉抗原。筛选出的特异性抗原蛋白进行质谱鉴定。通过以上方法,发现在western blotting反应中,有一个蛋白呈现出较强的特异性和灵敏度,经质谱鉴定为酸性磷酸酶(Acid phosphatase,Gi：238894261)蛋白。在NCBI基因组数据库中查询该蛋白的编码序列,设计引物扩增该基因,构建表达载体在大肠杆菌中诱导表达。表达蛋白经纯化后,以酶联免疫吸附试验(ELISA)对灵敏度和特异性进行验证,以阳性反应吸光度的平均值(A值)与交叉反应的A值之比确定信噪比。结果显示,该蛋白信噪比可达3.25,证明酸性磷酸酶为肺炎克雷伯杆菌的特异性抗原蛋白。嗜肺巴氏杆菌由于不能查到其基因组信息,尝试从其亲缘关系最近的菌种多杀巴氏杆菌中筛选特异性抗原。使用多杀巴氏杆菌总蛋白与嗜肺巴氏杆菌多抗反应筛选高灵敏度抗原,而与其他细菌多抗反应排除交叉抗原。结果显示,hypothetical protein PM1693 (Gi:15603558)呈现出较强的特异性和灵敏度,信噪比2.36,又由于小鼠不易感染多杀巴氏杆菌,该蛋白有望作为小鼠嗜肺巴氏杆菌检测的特异性抗原蛋白。
[Abstract]:Klebsiella pneumoniae (Klebsiella pneumonia), (Pasteurela pneumotropica) is a common pathogen in laboratory animals especially in mice and mice. China's national standards for laboratory animals: no specific pathogen (SPF) animals must be free of such bacterial infection. Nowadays, isolation and culture are the main methods for the detection of these two bacteria, which need to be improved because of their low sensitivity. The serological detection method based on specific antigen has the characteristics of high sensitivity, strong specificity, low cost, fast speed, and can be detected in the convalescent period after infection, so it is an excellent method for the detection of pathogenic infection in laboratory animals. In our study on the specific antigen of Klebsiella pneumoniae, the polyclonal antibodies were prepared by inoculating 10 common pathogens of mice inactivated by formaldehyde, and the total protein of Klebsiella pneumoniae was separated by two-dimensional electrophoresis. The protein antigen with strong immunogenicity was screened by immunoblot reaction of (western blotting) with polyclonal antibody of Klebsiella pneumoniae, and the cross antigen was excluded by reaction of total bacterial protein with other polyclonal antibodies. The specific antigen protein was identified by mass spectrometry. Through the above methods, we found that one protein showed strong specificity and sensitivity in western blotting reaction, and was identified as Acid phosphatase Giw 238894261 by mass spectrometry. The coding sequence of the protein was searched in the NCBI genomic database. Primers were designed to amplify the gene, and the expression vector was constructed to induce expression in Escherichia coli. After purification, the sensitivity and specificity of the expressed protein were verified by enzyme-linked immunosorbent assay (ELISA), and the signal-to-noise ratio (SNR) was determined by the ratio of the average absorbance of positive reaction (A value) to A value of cross-reaction. The results showed that the signal-to-noise ratio of the protein was 3.25, which proved that acid phosphatase was a specific antigen protein of Klebsiella pneumoniae. Because the genome information of Pasteurella pneumophilus could not be found, it was attempted to screen the specific antigen from Pasteurella multocida, the most closely related strain of Pasteurella pneumophilus. The high sensitivity antigen was screened by the reaction of the total protein of Pasteurella multocida with the polyclonal antibody of Pasteurella pneumophilus, but the cross antigen was excluded by the reaction with other bacteria. The results showed that hypothetical protein PM1693 (Gi:15603558) showed strong specificity and sensitivity, SNR 2.36, and that the protein could be used as a specific antigen protein for the detection of Pasteurella pneumophila in mice because it was not easy to infect Pasteurella multocida in mice.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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