mTOR特异性siRNA重组表达载体的构建及鉴定

目的构建mTORsiRNA重组表达载体，为探讨RNA干扰技术抑制巨噬细胞mTOR基因的相关研究奠定基础．方法设计具有短发夹结构的DNA序列，经退火成互补双链，再克隆至载体pGPU6／GFP／Neo中构建重组表达载体，转化DH5c~菌株，提取质粒行酶切鉴定后，并进行序列测定。再转染人巨噬细胞RAW264．7，进行荧光摄像和阳性克隆筛选。Westernblot方法检测巨噬细胞mTOR蛋白表达。结果DNA测序结果及PCR鉴定证实成功构建小鼠mTORsiRNA重组表达载体。Westernblot结果显示转染该重组载体的巨噬细胞mTOR蛋白表达下降约4l％。结论mTOR靶向RNA干扰重组表达载体构建成功，可以进行稳定筛选，该载体对巨噬细胞mTOR蛋白表达有抑制作用。

Construction and identification of mTOR specific siRNA recombinant expression vector

Objective To construct mTOR specific siRNA recombinant expression vector and provide foundation to inhibit macrophage mTOR gene by intenferene. Methods Short hairpin DNA sequence was annealed to form double chain, then cloned into vector pGPU6/GFP/Neo to construct the recombinant expression vector, and then transformed into DH5ct strain. Plasmid was identified by restriction enzyme digestion and sequencing, and transfeeted into macrophage RAW264.7. Fluorescence imaging and positive clones were screened after plasmid transfection. Expression of roTOR protein in maerophage was detected by western blot method. Results DNA sequencing and PCR analysis confirmed that mice roTOR siRNA recombinant expression vector was con- strueted successfully. Western blot results showed that the expression of mTOR protein in macrophage that be transfected with the recombinant vector decreased by 41%. Conclusion RNA interference targeting mTOR recombinant expression vector was success- fully constructed, and could be stabilized filter. The vector had inhibitory effect on mTOR expression in maerophage.