结核分枝杆菌PPE68基因真核表达载体的构建、表达和鉴定

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I-IeLa细胞中表达，Westernblot鉴定表达产物。结果：PCR产物、pGEM-T—PPE68及pcDNA3．1（＋）一PPE68分别经双酶切后均获得同一大小基因片段，表达产物经Westernblot鉴定相对分子质量约为40000。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。

Construction,expression and identification of the eukaryotic expression vector carrying Mycobacterium tuberculosis PPE68 gene

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.