【摘要】： 肺炎克雷伯桿菌(Klebsiella pneumonia),嗜肺巴氏桿菌(Pasteurela pneumotropica)是實(shí)驗(yàn)動(dòng)物特別是大小鼠的常見病原菌；實(shí)驗(yàn)動(dòng)物感染后將對實(shí)驗(yàn)結(jié)果造成影響。我國實(shí)驗(yàn)動(dòng)物國家標(biāo)準(zhǔn)中規(guī)定：無特定病原體(SPF)動(dòng)物必須無此類細(xì)菌感染。當(dāng)今對于這兩種細(xì)菌的檢測主要是分離培養(yǎng)法,該方法因靈敏度低有待改進(jìn)�；谔禺愋钥乖难鍖W(xué)檢測方法擁有靈敏度高,特異性強(qiáng),成本低,速度快,感染后恢復(fù)期的動(dòng)物也能檢出等特點(diǎn),是實(shí)驗(yàn)動(dòng)物病原感染檢測優(yōu)選方法。 我們在對肺炎克雷伯桿菌特異性抗原的研究中,采用甲醛滅活的10種小鼠常見病原菌菌體免疫小鼠制備多克隆抗體,使用雙向電泳分離肺炎克雷伯桿菌的總蛋白,并以免疫印跡法(western blotting)與肺炎克雷伯桿菌多克隆抗體反應(yīng),篩選出免疫原性強(qiáng)的蛋白抗原；再將細(xì)菌總蛋白與其他多克隆抗體反應(yīng)來排除交叉抗原。篩選出的特異性抗原蛋白進(jìn)行質(zhì)譜鑒定。通過以上方法,發(fā)現(xiàn)在western blotting反應(yīng)中,有一個(gè)蛋白呈現(xiàn)出較強(qiáng)的特異性和靈敏度,經(jīng)質(zhì)譜鑒定為酸性磷酸酶(Acid phosphatase,Gi：238894261)蛋白。 在NCBI基因組數(shù)據(jù)庫中查詢該蛋白的編碼序列,設(shè)計(jì)引物擴(kuò)增該基因,構(gòu)建表達(dá)載體在大腸桿菌中誘導(dǎo)表達(dá)。表達(dá)蛋白經(jīng)純化后,以酶聯(lián)免疫吸附試驗(yàn)(ELISA)對靈敏度和特異性進(jìn)行驗(yàn)證,以陽性反應(yīng)吸光度的平均值(A值)與交叉反應(yīng)的A值之比確定信噪比。結(jié)果顯示,該蛋白信噪比可達(dá)3.25,證明酸性磷酸酶為肺炎克雷伯桿菌的特異性抗原蛋白。 嗜肺巴氏桿菌由于不能查到其基因組信息,嘗試從其親緣關(guān)系最近的菌種多殺巴氏桿菌中篩選特異性抗原。使用多殺巴氏桿菌總蛋白與嗜肺巴氏桿菌多抗反應(yīng)篩選高靈敏度抗原,而與其他細(xì)菌多抗反應(yīng)排除交叉抗原。結(jié)果顯示,hypothetical protein PM1693 (Gi:15603558)呈現(xiàn)出較強(qiáng)的特異性和靈敏度,信噪比2.36,又由于小鼠不易感染多殺巴氏桿菌,該蛋白有望作為小鼠嗜肺巴氏桿菌檢測的特異性抗原蛋白。
[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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

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