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

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。

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 1 26 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 1 26 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.