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

作　　者：孟闯 万婷 徐正中 单法 解晓莉 申峻松 范峰 陈祥 焦新安 

机构地区：[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

出　　处：《畜牧兽医学报》2014年第5期789-794,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：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

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。The aim of this study was to express the fusion protein ESAT6-CFP10of 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-lhprecombinant plasmid.Fusion protein of ESAT6-CFP10was purified with His affinity chromatography column from strain of BL21(DE3)-pET30a(+)-esat6-linker-lhpafter 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-γassay for the detection of bovine tuberculosis.Results of SDS-PAGE and Western blotting impliedthat the fusion protein was expressed successfully and having good immunological reactivity. FACS analysis showed that there was a significant up-regulation of molecules both CD80and CD86expression on dendritic cells as well as CD69on splenic CD4+and CD8+T cells,meanwhile,more IFN-γspecific secreting cell spots rather than those of IL-4were detected by ELISPOT assay in C57BL/6mice which injected with the fusion protein.IFN-γtest results of 479cow blood samples showed that the fusion protein had the coincidence rates of 70.0%(positive)and 95.2%(negative)compared to IFN-γtest kit using PPD as stimulator.These data indicated the fact that this fusion protein had the capability of induce Th1immune response in mice and provided a basis for the diagnosis of bovine tuberculosis.

关 键 词：结核分枝杆菌 ESAT6 CFP10 细胞免疫 牛结核病 刺激原 

分 类 号：S852.61[农业科学—基础兽医学]