PTEN RNA干扰载体pSUPER的构建及功能鉴定  

Construction and Functional Identification of pSUPER Vector of PTEN RNAi

作　　者：陈健康[1] 何湘[2] 王莉[1] 刘元明[1] 杨晓莉[1] 

机构地区：[1]武警总医院检验科,北京100039 [2]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2016年第5期663-665,共3页Letters in Biotechnology

基　　金：武警部队课题(WZ2010016;WZ2011021)

摘　　要：目的:构建抑制PTEN基因表达的pSUPER RNA干扰(RNAi)载体pSUPER PTEN并鉴定其功能。方法:化学合成一对编码短发夹RNA序列,靶向细胞PTEN基因的寡核苷酸链,退火,克隆到经BglⅡ、HindⅢ双酶切的pSU-PER质粒上,构建重组RNAi质粒pSUPER PTEN,通过双酶切鉴定及测序分析验证构建效果;将构建正确的质粒转染肝癌HepG2细胞,免疫印迹和qPCR检测HepG2中PTEN的表达及其对AKT信号通路的影响。结果:双酶切鉴定及测序结果分析表明碱基成功插入pSUPER PTEN载体指定位点,同时序列一致;免疫印迹结果证明pSUPER PTEN载体可抑制HepG2细胞中PTEN的表达,并且提高AKT的磷酸化水平。结论:靶向PTEN的pSUPER PTEN载体构建成功,该载体能够特异性抑制PTEN基因的表达。Objective： To construct a p SUPER RNA interference（RNAi） vector that can inhibit PTEN gene ex-pression, and identify its function. Methods： A pair of oligonucleotides coding for short hairpin RNA and target-ing PTEN gene of cell were chemically synthesized and annealed and inserted into pSUPER plasmids digestedwith BglⅡ and HindⅢ to construct the recombinant p SUPER RNAi plasmid（pSUPER PTEN）. Recombinant plas-mid pSUPER PTEN was identified by enzyme digestion and sequencing analysis. And transfection of p SUPERPTEN into HepG2, Western blotting and qPCR were used to confirm the PTEN expression of HepG2 and its im-pact on AKT signal path. Results： Recombinant p SUPER-PTEN vector was certified by enzyme digestion and se-quencing analysis. Western blotting showed pSUPER PTEN vector can inhibit the PTEN protein upregulation ofHepG2, and improved the level of pAKT. Conclusion： The p SUPER RNAi vector targeting PTEN was successfullyconstructed and it can specifically inhibit PTEN gene expression.

关 键 词：PTEN PSUPER RNA干扰 PI3K/AKT 

分 类 号：Q78[生物学—分子生物学]