嗜肺军团菌重组蛋白PIP-Linker-MOMP的制备及其在血清学诊断中的研究

发布时间：2018-04-14 15:42

本文选题：嗜肺军团菌 + pip基因　；参考：《宁夏医科大学》2014年硕士论文

【摘要】：目的以嗜肺军团菌的pip基因和momp基因构建原核重组质粒pET-plm，诱导表达的PIP-Linker-MOMP(PLM)蛋白作为包被抗原，建立间接ELISA方法进行血清学检测，评价其在诊断嗜肺军团菌感染中的可行性及应用价值。方法以重组质粒pET-pip和pET-momp为模板，PCR扩增得到pip基因和momp基因，定向克隆至载体质粒pET-32a(+)中，构建重组质粒pET-plm，并经限制性内切酶、PCR及核酸测序分析鉴定。将构建的原核重组质粒pET-plm转化至大肠杆菌BL21内，经过IPTG的诱导表达，产物通过SDS-PAGE电泳进行鉴定分析。将纯化的重组蛋白PLM作为包被抗原，通过十字交叉连续稀释分析法筛选出最佳包被抗原的浓度，建立间接ELISA方法，检测32例健康人血清、58例军团病疑似患者中抗嗜肺军团菌抗体IgG、IgM、IgA水平，然后分别与德国DRG公司的Legionella ELISA试剂盒和美国RD公司Human LP IgG-AbELISA Kit/Human LP IgM-Ab ELISA Kit/Human LP IgA-Ab ELISA Kit检测结果进行配对设计资料的ROC曲线分析，以此来评价嗜肺军团菌重组蛋白PLM在军团病诊断中的可行性。结果扩增出了嗜肺军团菌723bp的pip基因和830bp的momp基因，构建的重组质粒pET-plm将其基因测序结果提交GeneBank经Blast比对,结果表明表达载体中插入的核酸序列与所选取嗜肺军团菌LP1型菌株的相应核酸序列具有98%的同源性,11个碱基位点发生改变，1个氨基酸位点发生改变，由于两种氨基酸同为疏水性氨基酸以及结构较相似，对蛋白质的空间结构和功能影响不大，对抗原表位基本没有影响。表达并纯化出了67KD左右的融合蛋白PLM，纯化的蛋白浓度达到728μg/mL。用以纯化蛋白PLM作为包被抗原建立的间接ELISA检测方法分别检测血清中Lp特异性IgG、IgM、IgA抗体。与DRG IgG/M/A试剂盒比较：灵敏度92.6%，特异度92.1%，一致性Kappa值0.821（P 0.05），ROC曲线下面积0.923。与ELISA试剂盒（RD）进行比较：IgG抗体灵敏度91.7%，特异度90.9%，一致性Kappa值0.784（P0.05），ROC曲线下的面积0.913；IgM抗体灵敏度90.6%，特异度93.1%，一致性Kappa值0.831（P0.05），ROC曲线下的面积为0.919；IgA抗体灵敏度88.9%，特异度92.1%，一致性Kappa值0.793（P0.05），ROC曲线下的面积0.905。结论成功构建了嗜肺军团菌重组质粒pET-plm，诱导表达并纯化出融合蛋白PLM，纯化蛋白PLM作为诊断抗原对军团病疑似患者血清和健康人血清进行诊断，显示出较好的特异度、灵敏度和一致性，为目前军团病的血清学诊断方法的改进提供了较好的参考价值，为军团病的血清学诊断试剂盒的研制奠定了基础。
[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.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R517.9

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