文章摘要
孙爱华,范兴丽,严杰.基于淋病奈瑟菌外膜蛋白pIA-pIB融合基因重组产物酶联免疫吸附试验的建立和应用[J].中华流行病学杂志,2008,29(3):271-276
基于淋病奈瑟菌外膜蛋白pIA-pIB融合基因重组产物酶联免疫吸附试验的建立和应用
Establishment and application of enzyme linked immunosorbent assay based on the outer membrane pIA-pIB fusion gene of Neisseria gonorrhoeae
收稿日期:2007-07-19  出版日期:2014-09-15
DOI:
中文关键词: 淋病奈瑟菌;融合基因;酶联免疫吸附试验
英文关键词: Neisseria gonorrhoeae;Fusion gene; Enzyme linked immunosorbent assay
基金项目:浙江省医药卫生科学研究基金资助项目(2004A018)
作者单位E-mail
孙爱华 浙江医学高等专科学校 310053 杭州  
范兴丽 浙江医学高等专科学校 310053 杭州  
严杰 浙江大学医学院病原生物学系 Med_bp@zju.edu.cn 
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中文摘要:
      目的 克隆淋病奈瑟菌外膜蛋白pIA、pIB基因并构建pIA-pIB融合基因及其原核表达系统以及建立基于rPIA-PIB的酶联免疫吸附试验(ELISA).方法 采用连接引物PCR构建pIA-pIB融合基因,按常规分子生物学方法构建其原核表达系统.采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和BioRad凝胶图像分析系统检测目的重组蛋白rPIA-PIB表达情况,Ni-NTA亲和层析法提纯rPIA-PIB.以rPIA-PIB为包被抗原建立检测淋病患者血清标本中rPIA和/或rPIB特异性IgG的ELISA,以rPIA-PIB抗血清为一抗建立检测淋病患者脓液标本中rPIA和/或rPIB的ELISA,实验中以rPIA、rPIB及其抗血清相关ELISA为对照.结果 pIA-pIB融合基因与原始核苷酸和氨基酸序列相似性均为100%.rPIA-PIB表达量为细菌总蛋白的29.8%,其提纯物SDS-PAGE后显示单一条带.rPIA-PIB-IgG-ELISA检测119例淋病患者血清标本的阳性率(98.3%)明显高于rPIA-IgG-ELISA(30.3%)或rPIB-IgG-ELISA(66.4%)(P<0.01).rPIA-PIB-ELISA检测119例淋病患者脓液标本的阳性率(91.6%)也明显高于rPIA-IgG-ELISA(27.7%)或rPIB-IgG-ELISA(62.2%)(P<0.01).结论 成功构建淋病奈瑟菌pIA-pIB融合基因及其原核表达系统;较之单一的rPIA或rPIB,rPIA-PIB作为淋病相关检测试剂盒抗原具有明显的优越性.
英文摘要:
      Objective To clone pIA and pIB genes of Neisseria gonorrhoeae, and to construct pIA-pIB fusion gene and its prokaryotic expression system,and to establish enzyme linked immunosorbent assay (ELISA) based on rPIA-PIB for detecting serum and pus samples from gonorrhea patients and to evaluate the sensitivity and specificity of the ELISA. Methods pIA一pIB fusion gene was constructed by polymerase chain reaction ( PCR ) using linking primers and a prokaryotic expression system of the fusion gene was constructed by using routine molecular biological methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) plus BioRad Gel Image Analyzer was used to measure the expression of the target recombinant protein rPIA-PIB. Ni-NTA affinity chromatography was performed to extract and purify rPIA-PIB. An ELISA by using rPIA-PIB as the coated antigen for detecting the specific IgG against rPIA and/or rPIB in gonorrhea patients' sera as well as another ELISA by using rPIA-PIB antiserum as the first antibody for detecting the rPIA and/or rPIB in gonorrhea patients' pus samples were established. In these experiments, ELISAs associated with rPIA, rPIB and their antisera were applied as the controls. Results 100%similarities of the nucleotide and putative amino acid sequences of the pIA一pIB fusion gene were confirmed when compared with the original sequences. The output of rPIA-PIB was 29.806 of the total bacterial proteins. The purified rPIA-PIB only showed a single target protein segment in gel after SDS- PAGE. Using a positive rate (98.3%)of rPIA-PIB-IgG-ELISA to detect 119 cases of gonorrhea patients' serum samples was remarkably higher than that of rPIA-IgG-ELISA(30.3)or rPIB-IgG-ELISA (66.4%)(P<0.01).The positive rate ( 91.6%)of rPIA-PIB-ELISA to detect 119 cases of gonorrhea patients' pus samples was also significantly higher than that of rPIA-IgG-ELISA (27.79'0)or rPIB-IgG-ELISA ( 62. 2 0,6 (P<0.01).Conclusion In this study we successfully constructed p1A一pIB fusion gene of N and its prokaryotic expression the antigen in gonorrhea associated detection kits system while rPIA-PIB showed obvious superiority used as compared to both the rPIA and rPIB.
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