插入四环素操纵子对恶性疟原虫血型糖蛋白结合蛋白130基因启动子活性的影响(英文)

Effects of Tetracycline Operator Element Insertion on Plasmodium falciparum Glycophorin Binding Protein 130 Gene Promoter Activity

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 )

Objective To construct tetracycline operator (TetO) modified glycophorin binding protein 130 gene (GBP130) promoter of Plasmodium falciparum and investigate the position effect of insertion of TetO on the promoter activity. Methods Cloning of 7-copy of TetO (7cot) sequence into 4 points relative to transcriptional initiation site of GBP130 promoter in pGBPCATΔ2 plasmid (2 upstream and 2 downstream), respectively, produced 4 derivative plasmids, pG/7T(-5), pG/7T(-2), pG/7T(+2) and pG/7T(+5). After transient transfection, the expression level of reporter gene CAT in both pGBPCATΔ2 and its derivative plasmids was detected and analysed by CAT ELISA. Results Identification by enzyme restrictions and PCR amplifications, as well as DNA sequencing confirmed that the plasmids were successfully constructed. Transfection of these plasmids and CAT detection suggested that insertion of 7cot into each point of GBP130 promoter enhanced the promoter activity, and downstream location of 7cot in the promoter showed higher promoter activity than those located upstream, with the strongest effect in Ins 4 (point +5). Conclusion Plasmid pG/7T(+5), in which 7cot was inserted into +5 point of GBP130 promoter, can be chosen as the responsive plasmid in establishing tetracycline-controlled transgenic expression system of malarial parasites.