p21RNAi的pSUPER载体的构建及功能鉴定

目的：构建抑制p21基因表达的pSUPERRNAi载体（pSUPER—p21）并鉴定其功能。方法：化学合成一对编码短发夹RNA序列的、靶向大鼠p21基因的寡核苷酸链，各60个碱基，退火，克隆到经BglⅡ、HindⅢ酶切的pSUPER质粒上，构建重组RNAi质粒（pSUPER-p21）。通过双酶切鉴定及测序分析验证构建效果。将正确构建的质粒转染大鼠原代培养皮质神经元，western blotting检测经红藻氨酸处理的神经元中p21蛋白表达。结果：pSUPER-p21载体经双酶切鉴定及测序分析，结果表明60个碱基成功插入到预计位点，并且序列完全一致。Western blotting结果证实pSUPER-p21载体可特异性抑制红藻氨酸诱导的原代培养皮质神经元中p21蛋白表达的上调。结论：靶向p21的pSUPERRNAi载体构建成功，该载体可特异性抑制p21基因表达。

Construction and Functional Identification of p SUPER Vector of p21 RNAi

Objective： To construct a pSUPER RNAi vector that can inhibit p21 gene expression and identify its function. Methods： A pair of 60bp oligonucleotides coding for short hairpin RNA and targeting p21 gene of rat were chemically synthesized and annealed and inserted into pSUPER plasmids digested with Bgl II and Hind HI to construct the recombinant pSUPER RNAi plasmid （pSU- PER-p21）. Recombinant pSUPER-p21 plasmid was identified by enzyme digestion and sequencing analysis. After transfection ofpSU- PER-p21 into primary cultured cortical neurons of rat, western blotting is used to determine the p21 protein expression of neurons treated with kainate（KA）. Results： Recombinant pSUPER-p21 vector was identified correct by enzyme digestion and sequencing analysis. West- em blotting showed pSUPER-p21 vector can inhibit the p21 protein up-regulation of primary cultured cortical neurons induced by KA. Conclusion： The pSUPER RNAi vector targeting p21 is successfully constructed and it can specifically inhibit p21 gene expression.