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.
出处
《国际流行病学传染病学杂志》
CAS
2011年第2期100-104,共5页
International Journal of Epidemiology and Infectious Disease
基金
浙江省科技厅承大项目(2007F10033)
浙江省医学科学院青年基金项目(A30810Q)