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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。
OBJECTIVE: To clone Mtb8.4 (MS), a new antigen of Mycobacterium tuberculosis, with signal peptide and construct its eukaryotic expression plasmid and identify it. Methods Mtb8.4 (MS) gene was amplified from the genome of Mycobacterium tuberculosis H37Rv by polymerase chain reaction (PCR) and digested with Hind Ⅲ and EcoRI and ligated with pcDNA3.1 (+) vector Connection and reorganization. Results After constructing pcDNA3.1 (+) - MS eukaryotic expression plasmid, the recombinant plasmid pcDNA3.1 (+) - MS was identified by restriction endonuclease digestion, PCR and DNA sequencing. Conclusion The successful construction of pcDNA3.1 (+) - MS eukaryotic expression plasmid, in order to further study the immune protective effect of the plasmid, to understand the signal peptide sequence in the MS protein expression and secretion of the role and preparation of the corresponding DNA vaccine for tuberculosis Foundation.