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目的构建抑制突变型p53基因siRNA表达载体。方法化学合成2段编码短发夹RNA序列的、靶向突变型p53基因的寡核苷酸(各58个碱基),退火,克隆到经BglⅡ、HindⅢ双酶切后的pSUPER-EGFP1(pSG)载体的polⅢH1启动子的下游,重组构建RNAi质粒,同时设立非特异对照。构建好的干扰载体瞬时转染胃癌细胞SGC-7901,用RT-PCR检测其对突变型p53基因的抑制效果。结果重组构建的pSUPER-EGFP1-p53(pSG-p53i)载体经双酶切电泳分析及插入基因序列分析,58个碱基成功插入到预计位点,并且序列完全一致。RT-PCR显示pSG-p53i对突变型p53基因的表达具有较好的瞬时抑制作用。结论载体的成功构建,有助于深入研究其对突变型p53基因的稳定抑制作用。体内合成siRNA的方法,可将RNAi技术用于细胞培养和哺乳动物研究。
Objective To construct a siRNA expression vector that inhibits mutant p53 gene. METHODS: Two oligonucleotides (each 58 bases) targeting the mutant p53 gene were synthesized by chemical synthesis of short hairpin RNA sequences, and annealed and cloned into pSUPER-EGFP1 (pSG) digested with BglII and HindIII. Downstream of the polIIIH1 promoter of the vector, RNAi plasmids were recombinantly constructed and non-specific controls were set up. The constructed interference vector was transiently transfected into gastric cancer cells SGC-7901, and its inhibitory effect on mutant p53 gene was detected by RT-PCR. Results The pSUPER-EGFP1-p53 (pSG-p53i) vector constructed recombinantly was analyzed by double enzyme electrophoresis and inserted into the gene sequence analysis. 58 bases were successfully inserted into the predicted sites and the sequences were identical. RT-PCR showed that pSG-p53i had a good transient inhibition of mutant p53 gene expression. Conclusion The successful construction of the vector is helpful to further study the stable inhibition of the mutant p53 gene. In vivo synthesis of siRNAs can be used in cell culture and mammalian studies.