结核分枝杆菌含信号肽的Mtb8.4基因的克隆及真核表达质粒的构建

摘要

[Objectives] To construct and identify the pcDNA3.1(+)-MS eukaryotic expression plasmid. [Methods] Extracted DNA from M. Tuberculosis was amplified by PCR and the target gene we got was cloned into the unique HindⅢ and EcoR Ⅰ cloning sites of pcDNA3.1(+). [Results] The accuracy of pcDNA3.1(+)-MS plasmid constructs was confirmed by a series of molecular biology techniques. [Conclusion] The construction of pcDNA3.1(+)MS provided the possibility for investigating immunogenicity of the recombinant plasmid, studying on the role of the signal peptide in the protein expression and excretion and preparing a new tuberculosis vaccine.%目的对结核杆菌新抗原-带信号肽的Mtb8.4(MS)进行基因克隆,构建其真核表达质粒并加以鉴定.方法采用聚合酶链反应(PCR)从结核分枝杆菌H37Rv株基因组中扩增出带信号肽的Mtb8.4(MS)目的基因,经HindⅢ和EcoRI消化后,与pcDNA3.1(+)载体进行连接重组.结果 pcDNA3.1(+)-MS真核表达质粒构建完成后,用限制性内切酶消化、PCR及DNA测序等多种方法进行鉴定,证实其构建成功.结论pcDNA3.1(+)-MS真核表达质粒的成功构建,为进一步研究该质粒的免疫保护效果,了解信号肽序列在MS蛋白表达和分泌过程中所起的作用及制备相应的结核病DNA疫苗奠定了基础.