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[目的]对结核杆菌MTC28基因进行克隆,构建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株基因组中扩增带信号肽MTC28目的基因,经NheI和EcoRⅠ消化后,与PCDNA3.1(+)载体进行连接重组。[结果]PCDNA3.1(+)-MTC28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(+)-MTC28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MTC28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] The MTC28 gene of M. tuberculosis was cloned, and the eukaryotic expression plasmid was constructed and identified. [Method] The target gene MTC28 with signal peptide was amplified from the genome of Mycobacterium tuberculosis H37Rv by polymerase chain reaction (PCR). After digested with NheI and EcoRI, it was ligated with PCDNA3.1 (+) vector. [Results] After construction of the eukaryotic expression plasmid pcDNA3.1 (+) - MTC28 was completed, it was identified by restriction endonuclease digestion, PCR and DNA sequencing. [Conclusion] The successful construction of PCDNA3.1 (+) - MTC28 eukaryotic expression plasmid, in order to further study the immune protective effect of this plasmid, to understand the function of signal peptide sequence in MTC28 protein expression and secretion process and to prepare the corresponding tuberculosis DNA vaccine laid the foundation.