结核分枝杆菌rdESAT-6抗原在耻垢分枝杆菌中的制备及其血清学诊断研究

doi:10.3969/j.issn.1673-8640.2014.06.007

检验医学 ›› 2014, Vol. 29 ›› Issue (6): 607-612.DOI: 10.3969/j.issn.1673-8640.2014.06.007

结核分枝杆菌rdESAT-6抗原在耻垢分枝杆菌中的制备及其血清学诊断研究

郭建¹,范齐文²,范小勇¹,马辉¹,钱雪琴¹,胡香南¹,吴文娟²

1. 上海市(复旦大学附属)公共卫生临床中心医学检验科,上海 201508;
2. 上海市东方医院南院医学检验科,上海 200123

收稿日期:2013-11-05 出版日期:2014-06-30 发布日期:2014-06-23
通讯作者: 吴文娟,联系电话:021-20334503。
作者简介:郭建,男,1983年生,硕士,技师,主要从事病原微生物快速诊断及耐药性研究。
基金资助:
上海市卫生局青年课题资助项目(2010Y018);国家自然科学基金(81201340)

Preparation and serology diagnosis research of recombinant Mycobacterium smegmatis expressing rdESAT-6 of Mycobacterium tuberculosis

GUO Jian¹,FAN Qiwen²,FAN Xiaoyong¹, MA Hui¹, QIAN Xueqin¹, HU Xiangnan¹, WU Wenjuan²

1. Department of Clinical Laboratory, Shanghai Public Health Clinical Center,Fudan University, Shanghai 201508, China;
2. Department of Clinical Laboratory, South Branch, East Hospital, Shanghai 200123, China

Received:2013-11-05 Online:2014-06-30 Published:2014-06-23

摘要/Abstract

摘要： 目的构建结核分枝杆菌(MTB)融合基因2×esat-6原核表达载体,利用耻垢分枝杆菌诱导表达、纯化早期分泌抗原ESAT-6重组二聚体(rdESAT-6),Western blot分析其抗原性,并评估其在结核病患者中的血清学诊断价值。方法以DNA疫苗HG856A₂为模板扩增2×esat-6基因,构建pMF41-2×esat-6原核表达载体,电转化耻垢分枝杆菌,诱导表达rdESAT-6蛋白,Western blot分析其抗原性。收集126例确诊的结核病患者和42名健康体检者的血清,用间接酶联免疫吸附试验(ELISA)和结核斑点金免疫渗滤试验(TB-DOT)检测血清结核抗体,评估rdESAT-6在结核血清学诊断中的价值。结果成功构建了pMF41-2×esat-6重组质粒,以包涵体形式表达了rdESAT-6蛋白,纯化的蛋白纯度在95%以上,可与小鼠抗ESAT-6血清发生特异性反应。126例结核病患者血清检测结果表明,rdESAT-6蛋白在MTB阳性组和阴性组患者血清学诊断的敏感性分别为79.75% (63/79)、61.70% (29/47),好于TB-DOT的62.03% (49/79)、44.68% (21/47)(P<0.05)。2种方法对42名健康体检者血清诊断特异性均为95.24% (40/42)。结论 MTB的融合蛋白rdESAT-6用于结核病血清学检测具有较好的敏感性和特异性,可作为结核病血清学诊断的优选抗原。

关键词: 结核分枝杆菌, ESAT-6, rdESAT-6, 融合蛋白, 免疫学诊断

Abstract:

Objective To construct the prokaryotic expression vectors containing Mycobacterium tuberculosis(MTB) fusion gene. Meanwhile, the inducible expression of fusion protein recombinant dimer ESAT-6 (rdESAT-6) was performed in the expression systems of Mycobacterium smegmatis. The fusion protein was purified, and the antigenic specificities of fusion protein was analyzed by Western blot. The significance of fusion proteins for the diagnosis of tuberculosis was evaluated. Methods The 2×esat-6 gene was amplified with DNA vaccine HG856A₂. The 2×esat-6 gene was then cloned into plasmid pMF41 for the construction of prokaryotic expression vector pMF41-2×esat-6. The recombinant plasmid pMF41-2×esat-6 was electroporated into Mycobacterium smegmatis. The target gene was induced to express fusion protein rdESAT-6. The antigenic specificities of purified fusion protein was analyzed by Western blot with mouse antiserum against rdESAT-6. The sensitivity and specificity of these fusion proteins for the diagnosis of tuberculosis were assayed by enzyme-linked immunosorbent assay (ELISA) and tuberculosis dot immunogold filtration assay (TB-DOT) test kit for 126 tuberculosis patients and 42 healthy controls. Results The prokaryotic expression vector pMF41-2×esat-6 was constructed successfully, and the related fusion protein was expressed in the form of inclusion body. The fusion protein was purified, and the purity of these proteins were great than 95%. The fusion protein was antigenic with ESAT-6 mouse antiserum. The sensitivities of the 126 tuberculosis patients and negative cases of serological diagnosis were 79.75% (63/79) and 61.70% (29/47), respectively, which were better than those of TB-DOT test kit [62.03% (49/79) and 44.68% (21/47),P<0.05] . The specificity was both 95.24% (40/42). Conclusions The fusion proteins of MTB are successfully prepared in this study with good sensitivity and specificity, which can be used for the diagnosis of tuberculosis.

Key words: Mycobacterium tuberculosis, ESAT-6, Recombinant dimer ESAT-6, Fusion protein, Immunology diagnosis

中图分类号:

R446.5

引用本文

郭建,范齐文,范小勇,马辉,钱雪琴,胡香南,吴文娟. 结核分枝杆菌rdESAT-6抗原在耻垢分枝杆菌中的制备及其血清学诊断研究[J]. 检验医学, 2014, 29(6): 607-612.

GUO Jian,FAN Qiwen,FAN Xiaoyong, MA Hui, QIAN Xueqin, HU Xiangnan, WU Wenjuan. Preparation and serology diagnosis research of recombinant Mycobacterium smegmatis expressing rdESAT-6 of Mycobacterium tuberculosis[J]. , 2014, 29(6): 607-612.

使用本文

0
/ / 推荐

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://www.shjyyx.com/CN/10.3969/j.issn.1673-8640.2014.06.007

https://www.shjyyx.com/CN/Y2014/V29/I6/607

参考文献

[1] World Health Organization.Global tuberculosis control[S] . WHO, 2012.
[2] Srensen AL, Nagai S, Hoven G, et al. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis[J] .Infect Immun, 1995, 63(5): 1710-1717.
[3] Bergstedt W, Tingskov PN, Thierry-Carstensen B, et al. First-in-man open clinical trial of a combined rdESAT-6 and rCFP-10 tuberculosis specific skin test reagent[J] . PLoS One, 2010, 5(6) :e11277.
[4] Arend SM, Franken WP, Aggerbeck H, et al. Double-blind randomized Phase I study comparing rdESAT-6 to tuberculin as skin test reagent in the diagnosis of tuberculosis infection[J] . Tuberculosis, 2008, 88(3): 249-261.
[5] Aggerbeck H, Madsen SM. Safety of ESAT-6[J] . Tuberculosis (Edinb), 2005, 86(5):363-373.
[6] Luo F, Sun X, Wang Y, et al. Ficolin-2 defends against virulent Mycobacteria tuberculosis infection in vivo, and its insufficiency is associated with infection in humans[J] . PLoS One, 2013, 8(9): e73859.
[7] Xu Z, Lu D, Zhang X, et al. Mycobacterium smegmatis in skin biopsy specimens from patients with suppurative granulomatous inflammation[J] . J Clin Microbiol, 2013, 51(3):1028-1030.
[8] Mishra S, Bhagavat R, Chandra N, et al. Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis[J] . Protein Expr Purif, 2012, 86(1):58-67.
[9] Fan XY, Ma H, Guo J, et al. A novel differential expression system for gene modulation in Mycobacteria[J] . Plasmid, 2009, 61(1):39-46.
[10] 郭建,范齐文,卢水华,等.肺外结核感染的快速分子检测和菌株分型方法的应用[J] .中华检验医学杂志, 2013, 36(7):620-624.
[11] 岳乔红,苏明权,杨军兰,等. 以结核分枝杆菌38 kD 重组蛋白为抗原的血清学诊断[J] . 中国生物制品学杂志, 2006, 19(6): 633-635.
[12] 吴雪琼,张俊仙,哈光平,等. 结核分枝杆菌katG 蛋白的表达和纯化及血清学诊断价值[J] . 中华检验医学杂志, 2003, 26(5): 310-311.
[13] 郭建,范小勇,马辉,等. 结核分枝杆菌ESAT-6重组二聚体的表达、纯化及其在血清学诊断中的初步应用[J] . 中国生物制品学杂志, 2009, 22(2):128-131.
[14] Harboe M, Oettinger T, Wiker HG, et al. Evidence for occurrence of ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG[J] . Infect Immun, 1996, 64(1): 16-22.
[15] Sebina I, Cliff JM, Smith SG, et al. Long-lived memory B-cell responses following BCG vaccination[J] . PLoS One, 2012, 7(12):e51381.
[16] Tsolaki AG, Nagy J, Leiva S, et al. Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model[J] . Mol Immunol, 2013, 54(3-4): 278-83.
[17] Fan X, Gao Q, Fu R. DNA vaccine encoding ESAT-6 enhances the protective efficacy of BCG against Mycobacterium tuberculosis infection in mice[J] . Scand J Immunol, 2007, 66(5): 523-528.
[18] Brandt L, Elhay M, Rosenkrands I, et al. ESAT-6 subunit vaccination against Mycobacterium tuberculosis[J] . Infect Immun, 2000, 68(2): 791-795.
[19] Li Z, Song D, Zhang H, et al. Improved humoral immunity against tuberculosis ESAT-6 antigen by chimeric DNA prime and protein boost strategy[J] . DNA Cell Biol, 2006, 25(1): 25-30.

相关文章 15

[1] 孙信林, 叶星明, 李倩, 赵丽君. 支气管镜检查联合痰TB-RNA、血清TB-SA检测诊断菌阴肺结核[J]. 检验医学, 2023, 38(9): 870-873.

[2] 缪星国, 叶慧, 苏菲菲. GeneXpert MTB/RIF检出量与结核分枝杆菌培养和利福平表型耐药的关系[J]. 检验医学, 2023, 38(9): 874-877.

[3] 茹浩浩, 陈连勇, 杨星, 陈涛, 闫双群, 许琳. Xpert MTB/RIF与结核分枝杆菌利福平耐药表型检测一致性分析[J]. 检验医学, 2023, 38(11): 1032-1035.

[4] 郭明日, 李玉明, 李妍, 孙海柏. 微阵列芯片法和多同源基因序列测序在非结核分枝杆菌菌种鉴定中的差异[J]. 检验医学, 2023, 38(10): 909-914.

[5] 徐镠粤, 廖庆华, 彭柯皓, 余美玲, 陈燕梅, 卓文基, 赖晓宇. 核酸适配体荧光探针法和液基夹层杯法检测结核分枝杆菌临床应用价值比较[J]. 检验医学, 2023, 38(10): 915-918.

[6] 杜晶辉, 黄自坤, 刘旻, 龚菁菁, 王丹, 刘基波, 刘旭. 液体培养基硝酸还原酶试验快速检测临床常用抗结核药物最小抑菌浓度和临界浓度效果评价[J]. 检验医学, 2022, 37(6): 568-576.

[7] 李华, 王伟亮, 谢贝, 杨瑜, 孟繁荣, 王楠, 刘志辉, 张言斌. 流式细胞术鉴定结核分枝杆菌利福平异质性耐药可行性分析[J]. 检验医学, 2022, 37(6): 577-582.

[8] 王晶晶, 蒋晓飞, 马逸珉, 王蓓. 眼部感染休斯顿分枝杆菌1例报道[J]. 检验医学, 2021, 36(9): 988-990.

[9] 郁晨蕾, 江渊, 李静, 王莉莉, 陈炜, 沈旭晖, 张阳奕. 细菌超声分散计数仪在结核分枝杆菌最小抑菌浓度检测中的应用[J]. 检验医学, 2021, 36(6): 650-655.

[10] 林小玲, 梁立锋, 吴影桃, 赵杰明, 蔡秋月. 云浮地区肺结核病患者结核分枝杆菌复合群耐药性分析[J]. 检验医学, 2021, 36(2): 194-197.

[11] 刘军, 刘涛, 戴碗琴, 王艺翔, 侯彦强. Xpert MTB/RIF技术在结核分枝杆菌利福平耐药性快速检测中的价值[J]. 检验医学, 2020, 35(9): 920-923.

[12] 王启源, 姬文兰, 虞秀锋. 嗅素蛋白4对卡介苗诱导巨噬细胞凋亡和细菌胞内生存率的影响[J]. 检验医学, 2020, 35(8): 818-822.

[13] 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.

[14] 池宏波, 杨英梅. 台州地区结核分枝杆菌分离及耐药特征分析[J]. 检验医学, 2020, 35(5): 455-457.

[15] 赵金云, 金法祥, 许文芳, 许鹏, 童丹. 绍兴地区非结核分枝杆菌肺部感染情况分析[J]. 检验医学, 2020, 35(2): 125-128.

编辑推荐

Metrics

阅读次数

全文

摘要

摘要

参考文献

相关文章

编辑推荐

Metrics

回顶部

结核分枝杆菌rdESAT-6抗原在耻垢分枝杆菌中的制备及其血清学诊断研究

Preparation and serology diagnosis research of recombinant Mycobacterium smegmatis expressing rdESAT-6 of Mycobacterium tuberculosis

RichHTML

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	孙信林, 叶星明, 李倩, 赵丽君. 支气管镜检查联合痰TB-RNA、血清TB-SA检测诊断菌阴肺结核[J]. 检验医学, 2023, 38(9): 870-873.
[2]	缪星国, 叶慧, 苏菲菲. GeneXpert MTB/RIF检出量与结核分枝杆菌培养和利福平表型耐药的关系[J]. 检验医学, 2023, 38(9): 874-877.
[3]	茹浩浩, 陈连勇, 杨星, 陈涛, 闫双群, 许琳. Xpert MTB/RIF与结核分枝杆菌利福平耐药表型检测一致性分析[J]. 检验医学, 2023, 38(11): 1032-1035.
[4]	郭明日, 李玉明, 李妍, 孙海柏. 微阵列芯片法和多同源基因序列测序在非结核分枝杆菌菌种鉴定中的差异[J]. 检验医学, 2023, 38(10): 909-914.
[5]	徐镠粤, 廖庆华, 彭柯皓, 余美玲, 陈燕梅, 卓文基, 赖晓宇. 核酸适配体荧光探针法和液基夹层杯法检测结核分枝杆菌临床应用价值比较[J]. 检验医学, 2023, 38(10): 915-918.
[6]	杜晶辉, 黄自坤, 刘旻, 龚菁菁, 王丹, 刘基波, 刘旭. 液体培养基硝酸还原酶试验快速检测临床常用抗结核药物最小抑菌浓度和临界浓度效果评价[J]. 检验医学, 2022, 37(6): 568-576.
[7]	李华, 王伟亮, 谢贝, 杨瑜, 孟繁荣, 王楠, 刘志辉, 张言斌. 流式细胞术鉴定结核分枝杆菌利福平异质性耐药可行性分析[J]. 检验医学, 2022, 37(6): 577-582.
[8]	王晶晶, 蒋晓飞, 马逸珉, 王蓓. 眼部感染休斯顿分枝杆菌1例报道[J]. 检验医学, 2021, 36(9): 988-990.
[9]	郁晨蕾, 江渊, 李静, 王莉莉, 陈炜, 沈旭晖, 张阳奕. 细菌超声分散计数仪在结核分枝杆菌最小抑菌浓度检测中的应用[J]. 检验医学, 2021, 36(6): 650-655.
[10]	林小玲, 梁立锋, 吴影桃, 赵杰明, 蔡秋月. 云浮地区肺结核病患者结核分枝杆菌复合群耐药性分析[J]. 检验医学, 2021, 36(2): 194-197.
[11]	刘军, 刘涛, 戴碗琴, 王艺翔, 侯彦强. Xpert MTB/RIF技术在结核分枝杆菌利福平耐药性快速检测中的价值[J]. 检验医学, 2020, 35(9): 920-923.
[12]	王启源, 姬文兰, 虞秀锋. 嗅素蛋白4对卡介苗诱导巨噬细胞凋亡和细菌胞内生存率的影响[J]. 检验医学, 2020, 35(8): 818-822.
[13]	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.
[14]	池宏波, 杨英梅. 台州地区结核分枝杆菌分离及耐药特征分析[J]. 检验医学, 2020, 35(5): 455-457.
[15]	赵金云, 金法祥, 许文芳, 许鹏, 童丹. 绍兴地区非结核分枝杆菌肺部感染情况分析[J]. 检验医学, 2020, 35(2): 125-128.