摘要
目的构建mTOR siRNA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DH5α菌株,提取质粒行酶切鉴定后,并进行序列测定。再转染入巨噬细胞RAW264.7,进行荧光摄像和阳性克隆筛选。Western blot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mTOR siRNA重组表达载体。Western blot结果显示转染该重组载体的巨噬细胞mTOR蛋白表达下降约41%。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。
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.
出处
《实验与检验医学》
CAS
2013年第6期521-523,共3页
Experimental and Laboratory Medicine
基金
国家自然科学基金(30901284)
江西省自然科学基金(2009GQY0203)
