摘要目的 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pET-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-10编码基因用疏水甘氨酸接头(Gly4Ser)3进行PCR扩增融合.构建TA克隆载体pMD 19-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Nde Ⅰ、Xho Ⅰ双酶切后将融合基因定向克隆入原核表达载体pET-23b(+).将构建正确的重组质粒pET-23b(+).ec转化E.coli BL21(DE3)pLysE,异丙基-β-D-硫代半乳糖苷(IPTG)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Westem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21 000,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.

abstractsObjective To construct esat-6-cfp-10 fusion gene (ec) and its expression vector pET-23b( + )-esat-6-cfp-10 [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 OP-10 of MTB was linked with the (Gly4Ser)3 linker by gene SOEing. Construction of TA cloning vector pMDR 19-T-esal-6-cfp-10 (pMDR 19-T-ec) was identified by PCR, restriction analysis and sequencing. After pMDR 19-T-ec was digested with double restriction enzymes (Nde Ⅰ 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. coli. 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.