结核分枝杆菌ESAT6-CFP10蛋白的融合表达及其细胞免疫学特性

doi:10.11843/j.issn.0366-6964.2014.05.016

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (5): 789-794.doi: 10.11843/j.issn.0366-6964.2014.05.016

结核分枝杆菌ESAT6-CFP10蛋白的融合表达及其细胞免疫学特性

孟闯¹，万婷¹，徐正中¹，单法¹，解晓莉¹，申峻松¹，范峰²，陈祥^1*，焦新安^1*

（1．扬州大学江苏省人兽共患病学重点实验室/江苏省动物重要疫病与人兽共患病防控协同创新中心，扬州 225009；2．无锡市动物疫病预防控制中心，无锡 214026）

收稿日期:2013-11-06 出版日期:2014-05-23 发布日期:2014-05-23
通讯作者: 焦新安，E-mail：jiao@yzu.edu.cn；陈祥，E-mail：chenxiang@yzu.edu.cn
作者简介:孟闯（1985-），男，汉族，江苏徐州人，博士研究生，主要从事人兽共患病研究，E-mail：mengchuangyzu@163.com
基金资助:
国家“973”项目（2012CB518805）；国家公益性行业科研专项（200903027）；江苏省农业三新工程（SXGC (2012) 402）；江苏高校优势学科建设工程资助项目

Fusion Expression of Mycobacterium tuberculosis ESAT6 and CFP10 Protein and Its Cellular Immunoproperties

MENG Chuang¹， WAN Ting¹， XU Zheng-zhong¹， SHAN Fa¹， XIE Xiao-li¹， SHEN Jun-song¹， FAN Feng²， CHEN Xiang^1*， JIAO Xin-an^1*

(1.Jiangsu Key Lab of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses，Yangzhou University，Yangzhou 225009， China; 2.Wuxi Center of Animal Disease Control and Prevention，Wuxi 214026， China)

Received:2013-11-06 Online:2014-05-23 Published:2014-05-23
Supported by:

摘要/Abstract

摘要：

本研究旨在原核表达结核分枝杆菌ESAT6-CFP10融合蛋白，分析其在小鼠模型中的细胞免疫应答特性，并评价其作为牛结核外周血IFN-γ释放试验特异性刺激原的能力。首先，通过构建pET30a(+)-esat6-linker-lhp重组质粒，BL21(DE3)中诱导表达ESAT6-CFP10融合蛋白并纯化，随后经细胞表面分子的FACS分析、ELISPOT试验等分析其在小鼠中的细胞免疫应答特性，同时作为刺激原用于奶牛IFN-γ释放试验，评价其用于牛结核病诊断的潜能。结果表明：SDS-PAGE显示目的蛋白成功表达，Western blotting证实其对ESAT6、CFP10单克隆抗体有良好免疫反应性。FACS结果显示其上调树突状细胞表面CD80和CD86分子以及小鼠脾CD4⁺和CD8⁺ T细胞表面CD69的表达，ELISPOT结果表明其诱导的特异性IFN-γ分泌细胞数显著高于IL-4分泌细胞数，显示其诱导产生Th1型免疫应答的能力。在479份奶牛外周血样本的γ-干扰素释放试验中，ESAT6-CFP10蛋白与PPD作为刺激原的阳性符合率为70.0%，阴性符合率为95.2%。本研究成功表达ESAT6-CFP10融合蛋白，其具备诱导Th1型免疫应答特性，在牛结核外周血γ-干扰素释放试验中可作为候选刺激抗原。

Abstract:

The aim of this study was to express the fusion protein ESAT6-CFP10 of Mycobacterium tuberculosis and evaluate its cellular immunoproperties in mice model as well as the detection potential for bovine tuberculosis.The fragment of esat6-linker-lhp was amplified using PCR and then constructed into the pET30a(+)-esat6-linker-lhp recombinant plasmid.Fusion protein of ESAT6-CFP10 was purified with His affinity chromatography column from strain of BL21(DE3)-pET30a(+)-esat6-linker-lhp after induced with IPTG.The purified fusion protein was then used to evaluate its cellular immunoproperties in mice model as well as the potential usage in IFN-γ assay for the detection of bovine tuberculosis.Results of SDS-PAGE and Western blotting implied that the fusion protein was expressed successfully and having good immunological reactivity.FACS analysis showed that there was a significant up-regulation of molecules both CD80 and CD86 expression on dendritic cells as well as CD69 on splenic CD4⁺ and CD8⁺ T cells，meanwhile，more IFN-γ specific secreting cell spots rather than those of IL-4 were detected by ELISPOT assay in C57BL/6 mice which injected with the fusion protein.IFN-γ test results of 479 cow blood samples showed that the fusion protein had the coincidence rates of 70.0% (positive) and 95.2% (negative) compared to IFN-γ test kit using PPD as stimulator.These data indicated the fact that this fusion protein had the capability of induce Th1 immune response in mice and provided a basis for the diagnosis of bovine tuberculosis.

中图分类号:

S852.618

孟闯，万婷，徐正中，单法，解晓莉，申峻松，范峰，陈祥，焦新安. 结核分枝杆菌ESAT6-CFP10蛋白的融合表达及其细胞免疫学特性[J]. 畜牧兽医学报, 2014, 45(5): 789-794.

MENG Chuang， WAN Ting， XU Zheng-zhong， SHAN Fa， XIE Xiao-li， SHEN Jun-song， FAN Feng， CHEN Xiang， JIAO Xin-an. Fusion Expression of Mycobacterium tuberculosis ESAT6 and CFP10 Protein and Its Cellular Immunoproperties[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(5): 789-794.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2014.05.016

https://www.xmsyxb.com/CN/Y2014/V45/I5/789

参考文献

［1］WORLD HEALTH ORGANIZATION.Global Tuberculosis Control 2011［M］.WHO，2011.
［2］RODWELL T C，MOORE M，MOSER K S，et al.Tuberculosis from Mycobacterium bovis in Binational Communities，United States［J］.Emerg Infect Dis，2008，14(6)：909-916.
［3］BRODIN P，ROSENKRANDS I，ANDERSEN P，et al.ESAT-6 proteins：protective antigens and virulence factors?［J］.Trends Microbiol，2004，12(11)：500-508.
［4］LEWIS K N，LIAO R，GUINN K M，et al.Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guérin attenuation［J］.J Infect Dis，2003，187(1)：117-123.
［5］申峻松，张辉，张成全，等.结核分枝杆菌CFP10单克隆抗体的研制与初步鉴定［J］.细胞与分子免疫学杂志，2009，25(12)：1143-1145.
［6］陈祥，刘静，苗晓青，等.禽病原性大肠杆菌tsh突变株的构建及分离株tsh基因的检测［J］.畜牧兽医学报，2007，38(5)：493-499.
［7］SAMBROOK J，RUSSELL D W.Molecular Cloning：a laboratory manual［M］.3rd ed.New York：Cold Spring Harbor Laboratory Press，2001.
［8］INABA K，INABA M,ROMANI N，et al.Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with GM-CSF［J］.J Exp med，1992，176(6)：1693-1702.
［9］WOOD P R，JONES S L.BOVIGAM：an in vitro cellular diagnostic test for bovine tuberculosis［J］.Tuberculosis (Edinb)，2001，81(1-2)：147-155.
［10］FINE P E.Variation in protection by BCG：implications of and for heterologous immunity［J］.Lancet，1995，346(8986)：1339-1345.
［11］BERTHET F X，RASMUSSEN P B，ROSENKRANDS I，et al.A Mycobacterium tuberculosis operon encoding ESAT6 and a novel low-molecular-mass culture filtrate protein (CFP10)［J］.Microbiology，1998，144(11)：3195-3203.
［12］RENSHAW P S，LIGHTBODY K L，VEVERKA V，et al.Structure and function of the complex formed by the tuberculosis virulence factors CFP10 and ESAT6［J］.EMBO J，2005，24(14)：2491-2498.
［13］WEINRICH OLSEN A，VAN PINXTEREN L A，MENG OKKELS L，et al.Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85B and ESAT6［J］.Infect Immun，2001，69(5)：2773-2778.
［14］AHMAD S.Pathogenesis，immunology，and diagnosis of latent Mycobacterium tuberculosis infection［OL/J］.Clin Dev Immunol，2011，2011：814943.http://www.hindawi.com/journals/jir/2011/814943.
［15］MIHRET A.The role of dendritic cells in Mycobacterium tuberculosis infection［J］.Virulence，2012，3(7)：654-659.
［16］FLYNN J L.Immunology of tuberculosis and implications in vaccine development［J］.Tuberculosis (Edinb)，2004，84(1-2)：93-101.
［17］张辉，潘志明，焦新安.一种细胞因子检测新方法-ELISPOT技术［J］.动物医学进展，2004，25(6)：29-31.
［18］GORMLEY E，DOYLE M B，FITZSIMONS T，et al.Diagnosis of Mycobacterium bovis infection in cattle by use of the gamma-interferon (Bovigam) assay［J］.Vet Microbiol，2006，112(2-4)：171-179.

[1]	于志瑞, 张旭, 牛莎莎, 邓光存, 吴晓玲. LncRNA NR_003508通过海绵吸附miR-483-3p并靶向MLKL调控BCG感染小鼠巨噬细胞坏死[J]. 畜牧兽医学报, 2022, 53(9): 3149-3159.
[2]	宋禹昊, 李东, 孙江洋, 时坤, 李健明, 宗颖, 赵丹, 曾范利, 杜锐. CRISPR/Cas系统及其在结核分枝杆菌研究中的应用[J]. 畜牧兽医学报, 2020, 51(11): 2613-2621.
[3]	王健宏, 徐兆坤, 李武. 炎性小体的激活机制及其在机体抗结核分枝杆菌中的作用研究进展[J]. 畜牧兽医学报, 2020, 51(1): 27-34.
[4]	徐兆坤, 李武, 王玉炯. 外泌体在机体抗结核分枝杆菌感染中的作用及其应用[J]. 畜牧兽医学报, 2018, 49(9): 1803-1809.
[5]	李强, 刘春法, 何小丽, 李凡飞, 魏凡华, 赵德明, 周向梅, 许立华. cGAS介导牛分枝杆菌诱导小鼠髓源树突状细胞成熟[J]. 畜牧兽医学报, 2017, 48(5): 914-921.
[6]	张妍，宋纪伟，曾范利，时坤，李健明，刘杨，刘菲，孙凡婷，杜锐. 不同毒力结核分枝杆菌对THP-1细胞凋亡及细胞因子表达的影响[J]. 畜牧兽医学报, 2015, 46(9): 1600-1605.

结核分枝杆菌ESAT6-CFP10蛋白的融合表达及其细胞免疫学特性

Fusion Expression of Mycobacterium tuberculosis ESAT6 and CFP10 Protein and Its Cellular Immunoproperties

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 6

编辑推荐

Metrics

本文评价