结核分枝杆菌CFP10-PPE68融合基因原核表达质粒的构建及表达

Construction and expression of prokaryotic expression vector for culture filtrate protein 10-pentose-5-phosphate-3-epimerase 68 fusion gene of Mycobacterium tuberculosis

目的构建结核分枝杆菌CFP10-PPE68融合基因原核表达质粒,并在大肠杆菌中表达融合蛋白。方法以结核分枝杆菌H37Rv株基因组DNA为模板,采用PCR法分别扩增CFP10和PPE68基因,基因拼接法扩增CFP10-PPE68融合基因,克隆至pET-32a(+)载体,构建重组表达质粒pET-32a(+)-CFP10-PPE68,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒pET-32a(+)-CFP10-PPE68经双酶切和测序证明构建正确;表达的融合蛋白相对分子质量为68 630,表达量为菌体总蛋白的41.8%,主要以可溶性形式存在,且可与PPE68 rBCG免疫兔血清发生特异性反应。结论成功构建了结核分枝杆菌CFP10-PPE68融合基因原核表达质粒,并在大肠杆菌BL21(DE3)中表达了融合蛋白,为其在结核病血清学诊断中的应用奠定了基础。

Objective To construct a prokaryotic expression vector for culture filtrate protein 10 （CFPlO）-pentose-5-phosphate- 3-epimerase 68 （PPE68） fusion gene of Mycobacterium tuberculosis and express in E. coll. Methods CFP10 and PPE68 genes were amplified by PCR respectively using the genomic DNA of M. tuberculosis H37Rv strain as template, based on which fusion gene CFP10-PPE68 was amplified by gene SOEing, and cloned into vector pET-32a （＋）. The constructed recombinant plasmid pET-32a （＋）-CFP10-PPE68 was transformed to E. coli BL21 （DE3） and induced with IPTG, and the expressed product was identified by SDS-PAGE and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pET-32a （＋）-CFPlO- PPE68 was constructed correctly. The expressed fusion protein, with a relative molecular mass of 68 630, contained 41.8% of total somatic protein, mainly existed in a soluble form, and showed specific reaction with sera of rabbits immunized with PPE68 rBCG. Conclusion The prokaryotic expression vector for CFP10-PPE68 fusion gene of M. tuberculosis was constructed successfully, and fusion protein was expressed in E. coli BL21 （DE3）, which laid a foundation of application of the fusion protein to serological diagnosis of tuberculosis.