c-myc靶向siRNA真核表达载体的构建和鉴定

EstabIishment and Identification of siRNA Eukaryotic Expression Vectors of Recombinant Targeting Gene c-myc

目的:设计构建重组体为转染肿瘤细胞,观察癌基因的沉默效果,为探索肿瘤基因治疗的新途径打好基础。方法:以c-myc为靶基因,以pEGFP-C1/U6质粒为载体,设计构建重组体,根据c-myc癌基因cDNA序列,设计有小发夹结构的3条寡核苷酸序列,克隆到空载体pEGFP-C1/U6中,转化DH5а菌株,提取质粒,进行酶切鉴定和测序分析。结果:经酶切鉴定筛出的重组体测序结果与目的序列相同,重组载体构建成功。结论:利用RNAi技术可成功构建小干扰RNA重组体。

Objective: To design and establish transfection tumor cells from recombinant, observe dumbness effect of oncogene, and explore the new method of gene therapy. Methods: Recombinant were designed and established by targeting gene c-myc and plasmid pEGFP-C1/U6 based on c-myc oncogene cDNA sequence. Three pairs of oligonucleotides were synthesized and inserted into plasmid pEGFP-C1/U6 to generate siRNA eukaryotic expression vectors. DH5a strains were transformed, plasmids were extracted, and the recombinant sequences were identified. Results: The result of recombinant sequence was the same as aim sequence. The recombinant vectors were established successfully. Conclusion: siRNA recombinant can be established successfully by RNAi technique.