抑制Smad3的pSUPER RNAi系统的构建和活性鉴定

目的:构建抑制Smad3的shRNA表达载体.方法:化学合成2段编码短发夹RNA序列的、靶向Smad3的寡核苷酸,各64个碱基,退火,克隆到经BgL Ⅱ和Hind Ⅲ双酶切同时经牛小肠碱性磷酸酶(alkaline phosphatase,CIP)处理后的pSUPER载体的poly Ⅲ H1启动子的下游,重组构建RNA干扰(RNA induced interference,RNAi)质粒,同时设立错义寡核苷酸作为非特异性对照.并将所构建的pSUPER Smad3转染到人类肾小管上皮细胞(human renal tubular epithelial cells,HKC),检测其抑制效果.结果:重组构建的pSUPER Smad3载体经双酶切电泳分析及插入基因片段序列分析,结果表明64个碱基成功插入到预计位点,并且序列完全一致.体外活性鉴定表明其能成功地抑制Smad3的基因和蛋白表达,却不影响Smad2的表达.结论:载体成功构建,其在体外抑制效率高,且特异性好.

Construction and activity evaluation of pSUPER RNAisystem that inhibits Smad3

OBJECTIVE: To construct the expressing vector of siRNA which can inhibit the Smad3 activity. METHODS: Sixty-four bases of 2 pair oligos for hairp in RNA expression which targeted Smad3 gene were chemically synthesized and annealed. pSUPER vector was linearized with BgL II and Hin d III treated with alkaline phosphatase (CIP). Anneled oligos were inserted into the downstream of the treated pSUPER's pol III H1 promoter to construct RNAi plasmid (pSUPER Smad3). Oligos with a scrambled sequence were used as a negative control. pSUPER Smad3 was transfected into human renal tubular epithelial cells (HKC). RESULTS: Recombinant pSUPER Smad3 vector was identified by the digestion with Eco R I and Hin d III, and confirmed by the sequencing analysis with T3 primer. Sixty-four bases had been inserted into the expected site. Furthermore, the insertion sequence was exactly corrected. The activity evaluation indicated that mRNA and protein of Smad3 but not Smad2 were inhibited by pSUPER Smad3 in HKC. CONCLUSION: The pSUPER Smad3 system has been constructed successfully, and has high inhibition and specificity in vitro.