结核杆菌含信号肽的Mtb8.4真核表达质粒的构建及鉴定

Construction of The pcDNA3.1(+)-MS Eukaryotic Expression Plasmid

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H 3 7Rv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。

Objective 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 EcoRⅠcloning sites of pcDNA3.1(+). Results The accuracy of pcDNA3.1(+)-MS plasmid constructs was confirmed by a series of molecularbiology techniques. Conclusion The construction of pcDNA3.1(+)-MS provided the possibility for investigating immunogenicity of the recombinant plasmid and studying on the role of the signal peptide in the protein expression and excretion, and preparation of a new tuberculosis vaccine.