本文選題：嗜肺軍團(tuán)菌 + pip基因��； 參考：《寧夏醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的以嗜肺軍團(tuán)菌的pip基因和momp基因構(gòu)建原核重組質(zhì)粒pET-plm，誘導(dǎo)表達(dá)的PIP-Linker-MOMP(PLM)蛋白作為包被抗原，建立間接ELISA方法進(jìn)行血清學(xué)檢測，評價其在診斷嗜肺軍團(tuán)菌感染中的可行性及應(yīng)用價值。 方法以重組質(zhì)粒pET-pip和pET-momp為模板，PCR擴(kuò)增得到pip基因和momp基因，定向克隆至載體質(zhì)粒pET-32a(+)中，構(gòu)建重組質(zhì)粒pET-plm，并經(jīng)限制性內(nèi)切酶、PCR及核酸測序分析鑒定。將構(gòu)建的原核重組質(zhì)粒pET-plm轉(zhuǎn)化至大腸桿菌BL21內(nèi)，經(jīng)過IPTG的誘導(dǎo)表達(dá)，產(chǎn)物通過SDS-PAGE電泳進(jìn)行鑒定分析。將純化的重組蛋白PLM作為包被抗原，通過十字交叉連續(xù)稀釋分析法篩選出最佳包被抗原的濃度，建立間接ELISA方法，檢測32例健康人血清、58例軍團(tuán)病疑似患者中抗嗜肺軍團(tuán)菌抗體IgG、IgM、IgA水平，然后分別與德國DRG公司的Legionella ELISA試劑盒和美國RD公司Human LP IgG-AbELISA Kit/Human LP IgM-Ab ELISA Kit/Human LP IgA-Ab ELISA Kit檢測結(jié)果進(jìn)行配對設(shè)計資料的ROC曲線分析，以此來評價嗜肺軍團(tuán)菌重組蛋白PLM在軍團(tuán)病診斷中的可行性。 結(jié)果擴(kuò)增出了嗜肺軍團(tuán)菌723bp的pip基因和830bp的momp基因，構(gòu)建的重組質(zhì)粒pET-plm將其基因測序結(jié)果提交GeneBank經(jīng)Blast比對,結(jié)果表明表達(dá)載體中插入的核酸序列與所選取嗜肺軍團(tuán)菌LP1型菌株的相應(yīng)核酸序列具有98%的同源性,11個堿基位點發(fā)生改變，1個氨基酸位點發(fā)生改變，由于兩種氨基酸同為疏水性氨基酸以及結(jié)構(gòu)較相似，對蛋白質(zhì)的空間結(jié)構(gòu)和功能影響不大，對抗原表位基本沒有影響。表達(dá)并純化出了67KD左右的融合蛋白PLM，純化的蛋白濃度達(dá)到728μg/mL。用以純化蛋白PLM作為包被抗原建立的間接ELISA檢測方法分別檢測血清中Lp特異性IgG、IgM、IgA抗體。與DRG IgG/M/A試劑盒比較：靈敏度92.6%，特異度92.1%，一致性Kappa值0.821（P 0.05），ROC曲線下面積0.923。與ELISA試劑盒（RD）進(jìn)行比較：IgG抗體靈敏度91.7%，特異度90.9%，一致性Kappa值0.784（P0.05），ROC曲線下的面積0.913；IgM抗體靈敏度90.6%，特異度93.1%，一致性Kappa值0.831（P0.05），ROC曲線下的面積為0.919；IgA抗體靈敏度88.9%，特異度92.1%，一致性Kappa值0.793（P0.05），ROC曲線下的面積0.905。 結(jié)論成功構(gòu)建了嗜肺軍團(tuán)菌重組質(zhì)粒pET-plm，誘導(dǎo)表達(dá)并純化出融合蛋白PLM，純化蛋白PLM作為診斷抗原對軍團(tuán)病疑似患者血清和健康人血清進(jìn)行診斷，顯示出較好的特異度、靈敏度和一致性，為目前軍團(tuán)病的血清學(xué)診斷方法的改進(jìn)提供了較好的參考價值，為軍團(tuán)病的血清學(xué)診斷試劑盒的研制奠定了基礎(chǔ)。
[Abstract]:Objective to construct a prokaryotic recombinant plasmid pET-plm from pip gene and momp gene of Legionella pneumophila, and to establish an indirect ELISA method for serological detection.To evaluate its feasibility and value in the diagnosis of Legionella pneumophila infection.Methods the pip gene and momp gene were amplified from recombinant plasmid pET-pip and pET-momp and cloned into vector plasmid pET-32a (). The recombinant plasmid pET-plm was constructed and identified by restriction endonuclease polymerase chain reaction (PCR) and nucleic acid sequencing.The constructed prokaryotic recombinant plasmid pET-plm was transformed into Escherichia coli BL21 and expressed by IPTG. The product was identified and analyzed by SDS-PAGE electrophoresis.The purified recombinant protein PLM was used as the coating antigen and the optimal concentration of the coating antigen was screened by cross cross continuous dilution method. The indirect ELISA method was established.The serum levels of IgGG antibody against Legionella pneumophila in 58 suspected patients with Legionnaires' disease were detected in 32 healthy persons.Then the ROC curves of matched design data were analyzed with Legionella ELISA kit of DRG company in Germany and Human LP IgG-AbELISA Kit/Human IgM-Ab ELISA Kit/Human IgA-Ab ELISA Kit of R & D company in USA.To evaluate the feasibility of Legionella pneumophila recombinant protein PLM in the diagnosis of Legionnaires disease.Results the pip gene of Legionella pneumophila 723bp and the momp gene of 830bp were amplified. The recombinant plasmid pET-plm was sequenced and submitted to GeneBank for Blast comparison.The results showed that the nucleic acid sequence inserted in the expression vector had 98% homology with the corresponding nucleic acid sequence of the selected Legionella pneumophila LP1 strain, 11 base sites changed and 1 amino acid site changed.Because the two amino acids are hydrophobic and similar in structure, they have little effect on the spatial structure and function of protein, but have no effect on antigenic epitopes.The fusion protein of 67KD was expressed and purified, and the concentration of purified protein reached 728 渭 g / mL.The indirect ELISA method for the detection of serum LP specific IgG MMA antibody was established by using the purified protein PLM as the coated antigen.Compared with the DRG IgG/M/A kit, the sensitivity was 92.6%, the specificity was 92.1%, and the consistent Kappa value was 0.821 (P 0.05). The area under the curve was 0.923.Compared with the ELISA kit, the sensitivity of ELISA antibody was 91.7, the specificity was 90.9, the consistent Kappa value was 0.784g P0.05A, the area under the ROC curve was 0.913. The sensitivity was 90.6, the specificity was 93.1g, the area under the consistent Kappa curve was 0.919, the area under the curve was 88.989, the specificity was 92.1g.The area under the Kappa curve of 0.793g P0.05a was 0.905.Conclusion Recombinant plasmids pET-plm of Legionella pneumophila were successfully constructed, and the fusion protein PLM was induced and purified. The purified protein PLM was used as diagnostic antigen to diagnose the serum of suspected patients with Legionnaires' disease and healthy people's serum.The sensitivity and consistency provided a good reference value for the improvement of serological diagnosis method of Legionnaires disease and laid a foundation for the development of serological diagnostic kit for Legionnaires disease.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R517.9

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