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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-l 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-l 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。
Objective: To construct eukaryotic expression vector of LEDGFp52 gene RNA interference (RNAi). Methods: Using LEDGFp52 as target gene and pGenSil-l plasmid as vector, the recombinant was designed and constructed. Based on the nucleotide sequence of LEDGFp52 provided by GenBank database, two hairpin-stranded nucleotides were designed according to the design principle of Tuschl The recombinant plasmid pGenSil-1 was transformed into empty vector pGenSil-1 and transformed into DH5α. The plasmid was extracted and identified by restriction enzyme digestion and sequencing analysis. Results: The sequence of the recombinants screened by restriction endonuclease digestion was exactly the same as the target sequence. The recombinant vector was successfully constructed. The recombinant plasmid was transfected into HeLa cells for 48h. The expression of LEDGFp52 protein was detected by Western blotting. Conclusion: Small interference RNA recombinants that inhibit the expression of LEDGFp52 can be successfully constructed using RNAi technology.