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目的 构建四环素操纵子 (TetO)修饰的恶性疟原虫血型糖蛋白结合蛋白 13 0基因 (GBP13 0 )启动子 ,并探讨TetO插入后对启动子活性影响的位置效应。 方法 将 7个拷贝的TetO ( 7cot)序列分别克隆入GBP13 0启动子转录起始点附近的 4个位点 ,上、下游各两个 ,产生 4个衍生质粒pG/ 7T( -5 ) ,pG/ 7T( -2 ) ,pG/ 7T( +2 )和pG/ 7T( +5 )。瞬时转染后通过CATELISA检测和分析pGBPCATΔ2和 4个衍生质粒中CAT报告基因的表达水平。 结果 限制性酶切、PCR扩增和DNA测序证明质粒构建成功。转染和CAT检测表明 ,7cot插入到GBP13 0启动子的 4个位点中均可提高启动子的活性 ,并且定位于启动子下游比定位于上游具有更高的活性 ,其中于 +5位点启动子活性最高。 结论 在疟原虫四环素诱导性转基因表达系统中质粒pG/ 7T( +5 )可作为反应质粒加以应用
Objective To construct a tetraploid operon (GPO) promoter of plasmodium falciparum glycoprotein binding protein 13 0 (GBP13 0) and to investigate the location effect of TetO on promoter activity. Methods Seven copies of TetO (7cot) were cloned into four sites near the transcription initiation site of GBP13 0, two upstream and downstream respectively. Four plasmids pG / 7T (-5), pG / 7T (-2), pG / 7T (+2) and pG / 7T (+5). The CAT reporter gene expression levels in pGBPCATΔ2 and 4 derived plasmids were detected and analyzed by CATELISA after transient transfection. Results Restriction enzyme digestion, PCR amplification and DNA sequencing proved that the plasmid was constructed successfully. Transfection and CAT assay showed that 7cot inserted into the GBP13 0 promoter increased the activity of the promoter in all 4 sites and located downstream of the promoter had higher activity than the upstream located in the +5 site The highest promoter activity. Conclusion Plasmid pG / 7T (+5) can be used as a reaction plasmid in the plasmodium tetracycline-inducible transgene expression system