抑制NGF表达的pSUPER-H1 RNAi系统的构建

Construction of pSUPER-H1 RNAi System to Inhibit the Expression of NGF

目的构建抑制神经生长因子活性的siRNA表达载体。方法构建含有PolⅢH1启动子的pSUPER-H1质粒,化学合成2对编码短发夹RNA的寡核苷酸序列,各64个碱基,退火、克隆到经BglⅡ、HindⅢ酶切处理的pSUPER-H1质粒PolⅢH1启动子下游,获得重组pSUPER-H1质粒,转化感受态大肠杆菌,挑选阳性克隆,抽取重组质粒,EcoRⅠ、HindⅢ双酶切电泳、测序鉴定。结果重组pSUPER-H1质粒成功转化感受态大肠杆菌,经EcoRⅠ、HindⅢ双酶切琼脂糖凝胶电泳分析,表明64个碱基的寡核苷酸成功插入到预计位点,经测序鉴定,表明序列完全一致。结论重组pSUPER-H1载体的成功构建,为进一步研究神经生长因子活性打下基础,同时开展了质粒介导在体内表达siRNA的方法。

Objective To construct the expressing vector of siRNA in order to inhibit nerve growth factor expression. Methods We constructed the plasmid that contained Pol Ⅲ H1 promotor （pSUPER-H1）. Two pair of oligonucleotides comprised of 64 bases were chemically synthetized and annealed, pSUPER-H1 vectors were linearized with Bgl Ⅱ and HindⅢ. The annealed oligonucleotides were inserted into downstream of H1 promotor to constructed the shRNA plasmid （pSUPER-H1- NGF）. Recombinant pSUPER-H1-NGF vectors were then transformed into competent E. coli. The positive clones were selected and recombinant pSUPER-H1-NGF plasmids were extracted. The plasmids were digested with EcoR Ⅰ and Hind Ⅲ and loaded in agarose gel electrophoresis. Results Recmbinant pSUPER-H1-NGF plasmids were transformed into competent E. coli. successfully. Agarose gel electrophoresis demonstrated that 64bp oligonucleotides were inserted into the expected sites. Sequencing analysis showed the inserted sequences were exactly correct. Conclusion Successful construction of recombitant pSUPER-H1-NGF plasmids provides the precondition of further study of NGF and develops the tools to express siRNA in vivo mediated by vectors.