FadD9重组蛋白在活动性肺结核诊断中的价值

doi:10.3969/j.issn.1673-8640.2020.06.020

摘要/Abstract

摘要：

目的探讨结核分枝杆菌（MTB）FadD9重组蛋白在活动性肺结核诊断中的价值。方法制备FadD9重组蛋白并进行鉴定,使用C57BL/6小鼠模型评估其免疫原性。以Ag85A蛋白为阳性参照抗原,检测145例活动性肺结核患者（活动性肺结核组）、38例结核患者密切接触者（MTB密切接触组）和101名体检健康者（正常对照组）血清样本中FadD9重组蛋白的特异性抗体水平,将FadD9重组蛋白作为抗原与58例肺结核患者、34例非肺结核患者的全血样本37 ℃共孵育20~24 h后,采用γ-干扰素释放试验（IGRA）检测,将检测结果与IGRA试剂盒中的标准管进行比对,并采用受试者工作特征（ROC）曲线分析FadD9重组蛋白诊断结核病的敏感性和特异性。结果 FadD9重组蛋白可在C57BL/6小鼠体内引起较高水平的体液免疫反应,且与阳性参照抗原Ag85A接近。活动性肺结核组抗FadD9抗体水平高于MTB密切接触组和正常对照组（P<0.000 1）,MTB密切接触组与正常对照组之间差异无统计学意义（P>0.05）。抗Ag85A抗体水平3组之间差异均无统计学意义（P>0.05）。ROC曲线分析结果显示,FadD9重组蛋白诊断结核病的曲线下面积（AUC）为0.682,最佳临界值为0.343,敏感性为96.8%,特异性为37.5%。结论 FadD9蛋白在活动性结核病的诊断中具有一定的临床应用潜力。

关键词: FadD9重组蛋白, γ-干扰素释放实验, 结核分枝杆菌

Abstract:

Objective To investigate the role of FadD9 recombinant protein of Mycobacterium tuberculosis（MTB） for the diagnosis of active tuberculosis. Methods FadD9 recombinant protein was prepared and identified. C57BL/6 mouse model was used to evaluate its immunogenicity. Using Ag85A protein as positive reference antigen,the specific antibody levels of FadD9 recombinant protein in serum samples of 145 active tuberculosis patients（active tuberculosis group）,38 close-contact tuberculosis patients（MTB close-contact group） and 101 healthy subjects（healthy control group） were determined. The FadD9 recombinant protein was incubated with blood samples of 58 tuberculosis patients and 34 non-tuberculosis patients at 37 ℃ for 20-24 h,and then was determined by interferon-gamma release assay（IGRA）. The results were compared with the standard tube in IGRA kit,and the sensitivity and specificity of FadD9 recombinant protein in the diagnosis of tuberculosis were analyzed by receiver operating characteristic（ROC）curve. Results FadD9 recombinant protein could induce a high level of humoral immune response in mice,which was close to that of Ag85A. Serum anti-FadD9 antibody level in active tuberculosis group was higher than those in healthy control group and MTB close-contact group（P<0.000 1）. There was no statistical significance between MTB close-contact group and healthy control group（P>0.05）. There was no statistical significance among the 3 groups in the level of anti-Ag85A antibody（P>0.05）. For the diagnosis of tuberculosis,the area under curve（AUC） of FadD9 recombinant protein was 0.682,the optimal cut-off value was 0.343,and the sensitivity and specificity were 96.8% and 37.5%,respectively. Conclusions FadD9 recombinant protein is potential in the diagnosis of active tuberculosis.

Key words: FadD9 recombinant protein, Interferon-gamma release assay, Mycobacterium tuberculosis

中图分类号:

R446.1

张君丽, 林晨, 王玉臣, 刘军, 袁俐, 潘稚芬, 张鹭. FadD9重组蛋白在活动性肺结核诊断中的价值[J]. 检验医学, 2020, 35(6): 614-619.

ZHANG Junli, LIN Chen, WANG Yuchen, LIU Jun, YUAN Li, PAN Zhifen, ZHANG Lu. Role of FadD9 recombinant protein for the diagnosis of active tuberculosis[J]. Laboratory Medicine, 2020, 35(6): 614-619.

图/表 6

图1 FadD9重组蛋白的制备和鉴定结果注：（a）FadD9重组蛋白的表达和纯化;M为相对分子质量Marker,1为未诱导的阴性对照,2为1 mmol/L IPTG诱导全菌表达,3为1 mmol/L IPTG诱导包涵体表达,4为1 mmol/L IPTG诱导可溶性表达;箭头所示为重组蛋白对应条带;（b）FadD9重组蛋白鉴定;M为相对分子质量Marker,1为FadD9重组蛋白

图2 FadD9免疫小鼠血清抗体水平注：Ag85A蛋白的阴性对照组PBS;FadD9重组蛋白的阴性对照组PBS;Ag85A蛋白;FadD9重组蛋白

图3 活动性肺结核组、MTB密切接触组及正常对照组FadD9重组蛋白和Ag85A蛋白特异性抗体水平的比较注：（a）抗FadD9抗体水平;（b）抗Ag85A抗体水平

图4 FadD9重组蛋白T细胞表位预测结果注：（a）FadD9强结合位点;（b）FadD9弱结合位点

表2 FadD9重组蛋白和IGRA标准管诊断肺结核的ROC曲线参数

项目	AUC	最佳临界值	敏感性/%	特异性/%
FadD9重组蛋白	0.682（0.507~0.858）	0.343	96.8	37.5
IGRA标准管	0.976（0.938~1.000）	0.935	93.5	100.0

图5 FadD9重组蛋白与IGRA标准管诊断肺结核的ROC曲线注：标准管;FaD9;参考线

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