结核分枝杆菌rESAT-6-CFP-10融合蛋白的构建和表达及其免疫反应性分析

Study on construction and expression and immunoreactivity of Mycobacterium tuberculosis recombinant ESAT-6- CFP-10

目的构建结核分枝杆菌H37Rv株esat．6-cfp-10（简称ec）融合基因及其原核表达载体pET-23b（＋）．esat．6-cfp-10[以下简称pE％23b（＋）-ec]，表达、纯化融合蛋白rESAT-6-CFP-10（简称rEC），并分析其免疫反应性。方法采用基因拼接技术将ESAT-6和CFP-IO编码基因用疏水甘氨酸接头（Gly4Ser），进行PCR扩增融合。构建TA克隆载体pMDR19-T-esat-6-cfp-10（简称pMD-19-T-ec），利用PCR、酶切和测序进行鉴定。经限制性内切酶NdeI、XhoI双酶切后将融合基因定向克隆入原核表达载体pET-23b（＋）。将构建正确的重导组质粒pET-23b（＋）-ec转化E-coliBL21（DE3）pLysE，异丙基-β-D-硫代半乳糖苷（IPm）诱导rEC的表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western印迹法分析其表达情况。用镍离子螯合亲和层析柱纯化融合蛋白。用Western印迹初步评价融合蛋白的免疫反应性。结果融合基因及其原核表达载体构建成功，融合蛋白以可溶性非包涵体形式表达，相对分子质量为21000，表达量约占菌体总蛋白的35％。经亲和层析后得到了纯度达97．2％的融合蛋白。Western印迹证实，融合蛋白能与确诊的肺结核患者血清发生特异性免疫应答。结论成功构建了原核表达载体pET-23b（＋）-ec，获得了rEC融合蛋白，为rEC融合蛋白在结核病诊断中的应用奠定了基础。

Objective To construct esat-6-cfp-10 fusion gene （ec） and its expression vector pET-23b（ ＋ ）-esat- 6-cfp-]O [pET-23b（ ＋ ）-ec], and express rESAT-6-CFP-10 （rEC）fusion protein of Mycobacterium tuberculosis （MTB） H37Rv and evaluate its immunoreactivity. Methods Fused gene encoding ESAT-6 and CFP-10 of MTB was linked with the （Gly4Ser）3 linker by gene SOEing. Construction of TA cloning vector pMD4 19-T-esat-6-cfp-lO （pMDR 19-T-ec） was identified by PCR, restriction analysis and sequencing. After pMDR 19-T-ec was digested with double restriction enzymes （Nde [ and Xho Ⅰ ）, the fused gene was inserted into prokaryotic expression vector pET-23b （ ＋ ）. The recombinant plasmid pET-23b（ ＋ ）-ec was transformed into E. coli Bl21 （DE3） pLysE, and IPTG was used to induce the expreestion of rEC. Its expression was detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot. rEC was purified by nickel-chelate affinity chromatography. The immunoreactivity of rEC was preliminarily evaluated by Western blot. Results A recombinant plasmid pET-23b （ ＋ ）-ec was successfully constructed and could stably express a 21 000 rEC, which accounted for 35% total proteins of bacillus. It existed in soluble form in E. coll. After purification, the purity of rEC reached 97.2%. Its immunoreactivity was confirmed by Western blot. Conclusions The prokaryotic expression vector pET-23b（ ＋ ）-ec has been constructed and the fusion protein rEC has been obtained successfully, which provides an experimental basis for the application of rEC in the diagnosis of tuberculosis.