重组结核分枝杆菌CFP10-ESAT-6融合蛋白的制备及应用研究  

Preparation and application of recombinant Mycobacterium tuberculosis CFP10-ESAT-6 fusion protein

作　　者：陈全[1] 朱道银[1] 骆旭东[1] 蒋英[1] 江山[1] 

机构地区：[1]重庆医科大学微生物学教研室,400016

出　　处：《中华结核和呼吸杂志》2004年第4期244-248,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：重庆市卫生局科学基金资助项目 (0 0 10 0 6)

摘　　要：目的　制备重组培养滤液蛋白 10和 6 0 0 0早期分泌性抗原靶的融合蛋白 (rCFP10 ESAT 6 ) ,研究其免疫学特性 ,评价其在结核病血清学诊断中的价值。方法　用基因拼接 (GeneSOEing)法扩增lhp ESAT 6融合基因、并克隆入pQE30质粒 ,表达、纯化rCFP10 ESAT 6蛋白 ,通过Westernblot分析其抗原性。建立豚鼠BCG免疫模型和结核分枝杆菌强毒株 (H3 7Rv)感染模型 ,建立以融合蛋白为抗原的酶联免疫吸附测定 (ELISA)法 ,检测豚鼠血清中的抗结核分枝杆菌抗体 ,并与以纯化蛋白衍生物 (PPD)为抗原的ELISA法比较。结果　重组质粒pQE30 CFP10 ESAT 6靶基因的测序结果与预计序列 (lhp linker ESAT 6 )完全一致。融合蛋白在DH5α菌中以可溶性非包涵体形式存在 ,表达量占菌体总蛋白的 4 0 % ,分子质量为 2 6 0 0 0 ,纯化后的蛋白纯度为 98% ,浓度为 1 2 g/L。Westernblot分析表明 ,融合蛋白与活动性肺结核患者血清、兔抗CFP10血清和兔抗ESAT 6血清都能发生特异性免疫反应。以 10只生理盐水处理豚鼠血清的平均吸光度 (A)值 + 2s为正常界限值 ,以融合蛋白为抗原 ,11份H3 7Rv感染豚鼠血清全部呈阳性反应 ,11份BCG免疫豚鼠血清仅 1份呈阳性反应 ;以PPD为抗原 ,11份H3 7Rv感染豚鼠血清全部呈阳性反应 ,11份BCG免疫豚鼠血清也?Objective To prepare the recombinant CFP10 ESAT 6 fusion protein, and to study its immunological characteristics, and its potential for serodiagnosis of tuberculosis Methods The lhp ESAT 6 fusion gene was amplified by Gene SOEing, and then cloned into pQE30 plasmid The recombinant CFP10 ESAT 6 fusion protein was expressed and purified Its antigenicity was confirmed by Western blot Animal models infected with M tuberculosis H 37 Rv strain and M bovis BCG respectively were made to evaluate the potential value of the fusion protein in the serodiagnosis of tuberculosis Results The sequence of recombinant plasmid pQE30 CFP10 ESAT 6 was identical to the predicted sequence The recombinant protein (rCFP10 ESAT 6), about 26 000, existed in the cytoplasm of DH5α in soluble form and represented 40% of the total bacterial protein The purity and concentration of the final product was 98% and 1 2 g/L, respectively Western blot showed that the rCFP10 ESAT 6 had good immunoreactivity with sera from patients with active tuberculosis and rabbits immunized with CFP10 and ESAT 6 respectively The positive cutoff value was A 490 plus 2 standard deviation from negative guinea pig sera detected by ELISA Serological reactivity to rCFP10 ESAT 6 was observed in 11 of the serum samples from guinea pigs with tuberculosis and 1 of sera from guinea pigs infected with BCG, while the serological reactivity to PPD was observed in 11 of sera from guinea pigs with tuberculosis and in 11 of sera from guinea pigs infected with BCG Conclusions The rCFP10 ESAT 6 fusion protein was highly expressed in soluble form in E coli It had antigenicity of both CFP10 and ESAT 6, and could be used to differentiate infection with M tuberculosis H 37 Rv strain from immunization with M bovis BCG The study provided experimental data for potential application of rCFP10 ESAT 6 in the diagnosis of tuberculosis

关 键 词：重组结核分枝杆菌 CFPlO-ESAT-6融合蛋白 制备 临床应用 

分 类 号：R446.6[医药卫生—诊断学]