[关键词]
[摘要]
设计并合成针对bcl-2 mRNA的“锤头型”(hammerhead)核酶基因,克隆后经测序表明序列正确。bcl-2和RZ基因经体外转录和切割实验后,表现出很强的切割活性,然后把RZ基因定向克隆于真核表达载体pDOR-neo,重组体被命名为pDOR-RZ。通过脂质体(lipofectin)介导的DNA转染法,成功地把pDOR-RZ导入HL-60细胞。用Southern印迹杂交、RNA斑点杂交和流式细胞仪(FCM),观察RZ基因在HL-60细胞内的表达。结果表明:(1)RZ在转染HL-60细胞后72小时得以表达;(2)由于RZ的表达,细胞内Bcl-2蛋白合成受到抑制;(3)在FCM图谱上可见到明显的凋亡峰。
[Key word]
[Abstract]
A hammerhead RZ DNA was designed and synthesized, which can specifically cleave the bcl-2 mRNA. After demonstration of right sequences by sequencing and cleavage activity of RZ by in vitro cleaving experiment, The RZ DNA was recombinated into the pDOR - neo vector to form the recombinant pDOR - RZ. Using lipofectin - mediated DNA transfectionpDOR-RZ was successfully introduced into HL - 60 cells. The RZ expression was observed by Southern, RNA dot blot hybridization and flow cytometry (FCM) . The results demonstrated that (a) the RZ was expressed in 72 hours after transfection; (b) the synthesis of Bel - 2 protein was inhibited by the expression of RZ; (c) apoptotic peak appeared in FCM.
[中图分类号]
[基金项目]
国家自然科学基金(No.39570790)
