文章摘要
罗巧,李霜君,肖彤洋,李马超,刘海灿,楼永良,万康林.结核分枝杆菌4种新抗原的克隆表达及血清学评价[J].中华流行病学杂志,2018,39(4):514-518
结核分枝杆菌4种新抗原的克隆表达及血清学评价
Cloning expression and serological evaluation on Mycobacterium tuberculosis four new antigens
收稿日期:2017-09-12  出版日期:2018-04-18
DOI:10.3760/cma.j.issn.0254-6450.2018.04.026
中文关键词: 结核分枝杆菌  抗原  免疫学诊断  酶联免疫吸附试验
英文关键词: Mycobacterium tuberculosis  Antigen  Immunological diagnosis  ELISA
基金项目:国家科技重大专项(2013ZX10003006-002-001)
作者单位E-mail
罗巧 325035 温州医科大学检验医学院生命科学学院  
李霜君 325035 温州医科大学检验医学院生命科学学院  
肖彤洋 102206 北京, 中国疾病预防控制中心传染病预防控制所 传染病预防控制国家重点实验室 感染性疾病诊治协同创新中心  
李马超 102206 北京, 中国疾病预防控制中心传染病预防控制所 传染病预防控制国家重点实验室 感染性疾病诊治协同创新中心  
刘海灿 102206 北京, 中国疾病预防控制中心传染病预防控制所 传染病预防控制国家重点实验室 感染性疾病诊治协同创新中心  
楼永良 325035 温州医科大学检验医学院生命科学学院  
万康林 325035 温州医科大学检验医学院生命科学学院
102206 北京, 中国疾病预防控制中心传染病预防控制所 传染病预防控制国家重点实验室 感染性疾病诊治协同创新中心 
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中文摘要:
      目的 初步评价结核分枝杆菌4种新抗原Rv0432、Rv0674、Rv1566c和Rv1547的血清学诊断价值。方法 以结核分枝杆菌实验室标准参照菌株H37Rv全基因组DNA为模板PCR扩增Rv0432、Rv0674、Rv1566c基因的完整序列,Rv1547基因分为两段(Rv1547-1Rv1547-2)扩增,与PET-32a表达载体构建重组质粒,重组蛋白利用亲和层析的方法进行纯化。待检测血清和BL21(DE3)菌体蛋白孵育进行预处理。各重组抗原用ELISA对151份待检血清(41份健康组血清和110份细菌学阳性结核患者组血清)进行IgG抗体检测。检测结果用受试者工作特征曲线对其诊断效能进行分析和评价。采用t检验比较目的蛋白在结核患者组和健康组的差异性。结果 成功克隆表达和纯化了蛋白Rv0432、Rv0674、Rv1566c、Rv1547-1和Rv1547-2,ELISA结果显示Rv0432、Rv0674、Rv1566c、Rv1547-1和Rv1547-2的敏感性、特异性、阳性预测值、阴性预测值、约登指数和曲线下面积分别为43.64%~92.73%、80.49%~92.68%、0.92~0.94、0.38~0.80、0.363~0.732和0.649~0.915。目的蛋白在结核组检测到的IgG抗体水平均大于健康组(P<0.000 1)。结论 结核分枝杆菌新抗原Rv0432、Rv0674、Rv1566c、Rv1547-1和Rv1547-2具有良好的血清学检测价值,可作为结核病免疫学诊断的候选抗原。
英文摘要:
      Objective To evaluate the serological diagnostic value of Mycobacterium (M.) tuberculosis four new antigens Rv0432, Rv0674, Rv1566c and Rv1547. Methods Rv0432, Rv0674, Rv1566c and Rv1547 were amplified from M. tuberculosis strain H37Rv genomic DNA by using PCR, among which Rv1547 was divided into two segments for amplification (Rv1547-1 and Rv1547-2). The segments were cloned into expression vector PET-32a while the recombinant proteins were purified by affinity chromatography. Serums were incubated with BL21 (DE3) proteins. Antibodies IgG against M. tuberculosis were tested with 151 serum samples (41 healthy people and 110 TB patients) by using ELISA. The diagnostic efficiency of antigens was analyzed by means of receiver operating characteristic curve. Difference of the objective proteins in TB patients and healthy controls was compared by t-test. Results Recombinant antigens Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2 were successfully expressed and purified. Results from ELISA showed that the sensitivity, specificity, positive predictive value, negative predictive value, Youden index and area under the curve of Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2, as 43.64%-92.73%, 80.49%-92.68%, 0.92-0.94, 0.38-0.80, 0.363-0.732 and 0.649-0.915. All the objective proteins showed significantly higher antibody levels in TB patients, when compared to the healthy controls (P<0.000 1). Conclusion The newly identified antigens Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2 all performed well when being used for TB serological diagnosis, thus were expected to be new candidate antigens used for TB diagnosis.
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