摘要
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 expressing vector of siRNA to inhabit mutant p53 gene activity. Methods Two ohgonucleotides containing 58 base-pair of hairpin RNA targeting mutant p53 gene were chemically synthesized and anne, ated. pSUPER-EGFP1 （pSG） was linearized with Bgl Ⅱ and HindⅢ, then the annealed oligonucleotides were inserted into the downstream of pol ill H1 promoter of pSG to construct RNAi plasmid. The constructed RNAi plasmid was transfected into SGC-7901 cells and RT-PCR was used to evaluate the RNA interference efficiency. Oligos with a scrambled sequence were used as a negative control. Results Recombinant pSUPER-EGFPl-p53 （pSG-p53i） was identified by digestion with EcoR Ⅱ and HindⅢ and confirmed by DNA sequencing analysis. The results demonstrated that the 58 bp had been inserted the expected site and the sequence was exactly correct. RT-PCR suggested that the expression of mutant p53 gene was inhabited by pSC,-p53i. Conclusion Mutant p53 RNAi system has been successfully constructed.
出处
《浙江医学》
CAS
2007年第10期1026-1028,共3页
Zhejiang Medical Journal
基金
浙江省自然科学基金资助项目(X20506)
