结核分枝杆菌PPE68基因真核表达载体的构建、表达和鉴定
Construction，expression and identification of the eukaryotic expression vector carrying Mycobacterium tuberculosis PPE68 gene
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摘要: 目的:建立结核分枝杆菌PPE68蛋白基因重组质粒的真核表达载体,为以后对PPE重组蛋白的免疫原性分析奠定基础。方法:提取结核分枝杆菌总DNA,PCR法扩增出PPE68编码基因,通过线性T克隆载体pGEM-T连接,亚克隆至真核表达载体pcDNA3.1(+)后,转染至HeLa细胞中表达,Western blot鉴定表达产物。结果:PCR产物、pGEM-T-PPE68及pcDNA3.1(+)-PPE68分别经双酶切后均获得同一大小基因片段,表达产物经Western blot鉴定相对分子质量约为40 000。结论:成功构建了结核分枝杆菌PPE68基因真核表达载体,并获得了表达产物。Abstract: Objective:To establish the eukaryotic expression vector of recombinant PPE68 protein in Mycobacterium tuberculosis gene plasmid and lay the foundation for the later analysis of immunogenicity of PPE recombinant protein． Methods:The total DNA of Mycobacterium tuberculosis was extracted and PPE68 gene was amplified by PCR． The DNA fragment was inserted into pGEM-T，then the target gene was subcloned into pcDNA3． 1( + ) after the DNA fragment was cut from pGEM-T． New recombinant plasmid was transfected into hela cells，then expression product was confirmed by Western blot． Results:The size of the PCR product was 1 104 bp， the inserts of pGEM-T-PPE68 and pcDNA3． 1( + )-PPE68 digested by HindIII and EcoRI were as long as PCR product． The product that recombinant DNA be expressed in HeLa cells was about 40 000． Conclusions:The Results demonstate eukaryotic expression vector carring PPE68 gene has been set up and the expression products has been obtained and appraised．
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