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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 the prokaryotic expression plasmid of Mycobacterium tuberculosis CFP10-PPE68 fusion gene and express the fusion protein in E. coli. Methods Genomic DNA of Mycobacterium tuberculosis strain H37Rv was used as a template to amplify the CFP10 and PPE68 genes by PCR. The CFP10-PPE68 fusion gene was amplified by gene splicing and cloned into pET-32a (+) vector to construct the recombinant plasmid pET -32a (+) - CFP10-PPE68 was transformed into E. coli BL21 (DE3) and induced by IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results The recombinant plasmid pET-32a (+) - CFP10-PPE68 was proved to be correct by double enzyme digestion and sequencing. The relative molecular mass of the expressed fusion protein was 68 630 and the expression level was 41.8% of the total bacterial proteins, mainly in soluble form Exists, and can react specifically with PPE68 rBCG-immunized rabbit serum. Conclusion The prokaryotic expression plasmid of Mycobacterium tuberculosis CFP10-PPE68 fusion gene was successfully constructed and the fusion protein was expressed in Escherichia coli BL21 (DE3), which laid the foundation for its application in serodiagnosis of tuberculosis.