PCR法扩增NOGO基因RNA干涉小发夹和体外效应研究

NOGO gene RNA interference small hairpin amplified by PCR and its effect in vitro

目的通过PCR方法获得带有U6启动子的NOGO基因RNA干涉小发夹。方法设计特异性引物,人工合成U6启动子的上游引物和带有NOGO-66反相互补序列的下游引物。PCR扩增带有U6启动子的小发夹,PCR产物与载体连接。酶切产物以1%琼脂糖凝胶进行电泳分析,释放出430bp基因片段的质粒初步确定为阳性重组质粒,并测序。结果成功地扩增了靶向NOGO基因RNAi的带有U6启动子的小发夹。结论PCR方法可快速简单、廉价地获得shRNA。

Objective To obtain NOGO gene RNA interference （RNAi） small hairpin human involving U6 promotor by PCR. Methods The specific primers were designed, up primers including human U6 promoter and down primers including NOGO reverse complement target sequence were synthesized. Small hairpin RNA （shRNA） including human U6 promoter was directly amplified by PCR. PCR products can be cloned with T-vector. Digestion production was analyzed by 1% agarose gel electrophoresis. The plasmid 430 bp gene fragment was verified as positive recombinant plasmid to be sequenced. Results NOGO gene RNAi shRNA including human U6 promotor was amplified successfully. Conclusions The shRNA can be amplified quickly, easily and cheaply by PCR.