一种高效表达小干扰RNA载体的构建研究

目的：用mir30前体骨架来构建高效表达小干扰RNA载体。方法：通过基因合成的方法，将mir30前体骨架进行全长基因合成，并在合成的mir30前体骨架中引入合适的酶切位点用于外源发夹DNA的克隆，然后将之克隆到含U6启动子的表达载体中。将针对绿色荧光蛋白（green fluorescent protein，GFP）的小干扰RNA克隆到以上表达载体中，通过转染及Western blot来检测新的小干扰RNA表达载体的基因沉默效率。结果：同目前常用的含polⅢ的小干扰RNA表达载体相比较，用mir30前体骨架表达针对GFP的小干扰RNA的表达载体可以明显抑制GFP的表达。结论：用mir30前体骨架构建的小干扰RNA表达载体可高效表达外源小干扰RNA。

Construction of an expression vector for high expression of siRNA

AIM:To construct the vector for efficient expression of siRNA using pre-mir30 backbone.METHODS:By chemical synthesis method,pre-mir30 backbone introduced an appropriate restriction enzyme site for foreign shRNA inserting was cloned into an expressing vector containing U6 promoter.The silencing efficiency of a new siRNA expressing vector was detected by transfection and Western blot.RESULTS:The new vector containing pre-mir30 backbone expressing siRNA against GFP could markedly inhibit the expression of GFP compared with the vector expressing control siRNA.CONCLUSION:siRNA expressing vector constructed by pre-mir30 backbone could highly express foreign siRNA.