AIM: To construct and identify LEDGFp52 eukaryotic expression vector for RNA interference.METHODS: Recombinants were designed and established by targeting gene LEDGFp52 and plasmid pGensil-1 based on LEDGFp52 cDNA sequences of Genomes. Two pairs of oligonucleotides were synthesized according to the Tuschl principle and inserted into plasmid pGenSil-I to generate siRNA eukaryotic expression vector. DH5α strains were transformed, plasmids were extracted, and recombinant vectors were identified by the restriction map and the sequence analysis. The cultured cells were transfected by the recombinant plasmid (pGensil-1-RNA. LEDGFp52-1). At 48 hours after transfection, the whole cell protein was extracted, and the protein level was detected using Western blotting with mouse anti-human LEDGFp52 monoclonal antibody.RESULTS: Recombinant plasmids completely concord with the designs by the restriction map and the sequence analysis,the proteinlevel of LEDGFp52 was down regulated at 48hours after transfecting pGensil-1- LEDGFp52-1 expression vector into HeLa cells, the recombinant eukaryotic expression vectors were successfully constructed.CONCLUSION: siRNA recombinant can be successfully constructed by RNAi technique to inhibit the expression of LEDGFp52.%目的:构建LEDGFp52基因RNA干扰(RNAi)的真核细胞表达载体.方法:以LEDGFp52为靶基因,以pGenSil-1质粒为载体,设计构建重组体,根据GenBank数据库提供的LEDGFp52基因核苷酸序列,按照Tuschl设计原则,选择设计两条带发夹结构的核苷酸序列,克隆到空载体pGenSil-1中,转化DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析.结果:经酶切鉴定筛选出的重组体测序结果与目的序列完全一致,重组载体构建成功,重组质粒转染HeLa细胞48h,Western blotting检测到LEDGFp52蛋白表达的改变.结论:利用RNAi技术可成功构建抑制LEDGFp52表达的小干扰RNA重组体.
展开▼
