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目的构建多发性骨髓瘤(MM)特异的 APE1siRNA 表达载体 pSilencer K-IE-IgP-APE1siRNA,观察其对 MM 细胞 APE1蛋白表达的特异性敲除作用。方法设计合成的 APE1siRNAcDNA 序列与线性化 pSilencer2.0-U6母本质粒连接后,用 BamH Ⅰ、EcoR Ⅰ双酶切,插入 IgP 寡核苷酸片段;随后用 EcoR Ⅰ酶切 pSilencer IgP-APE1siRNA,将线性化载体与回收 IEcDNA 片段用 T4 DNA 连接酶连接;再用 Xho Ⅰ酶切,与回收 Kappa cDNA 片段连接,克隆出 MM 特异表达载体 pSilencer K-IE-IgPAPE1siRNA,每次连接后均经酶切鉴定和测序确认。用脂质体转染法将重组质粒转染 KM3、HOS 和MDA-231细胞,Western blot 分析其对 KM3细胞 APE1的特异性敲除作用。结果 pSilencer K-IE-IgP-APE1siRNA 能特异且有效地敲除 KM3细胞 APE1蛋白表达,转染 siRNA 后培养2d 的 KM_3细胞 APE1相对表达水平为0.118±0.047,而单独脂质体转染组,APE1相对表达水平为0.988±0.029,基因缄默效率为90%。结论成功构建了针对 MM 的 APE1siRNA 表达载体。
Objective To construct specific siRNA expression vector pSilencer K-IE-IgP-APE1 for multiple myeloma (MM) and to observe the specific knockdown of APE1 protein in MM cells. Methods After the APE1siRNA cDNA sequence was designed and ligated with the linearized pSilencer2.0-U6 maternal plasmid, it was digested with BamH Ⅰ and EcoR Ⅰ to insert IgP oligonucleotide fragment. Subsequently, pSilencer IgP-APE1siRNA was digested with EcoR Ⅰ The linearized vector and the recovered IEcDNA fragment were ligated with T4 DNA ligase. After digested with Xho I and ligated with the recovered Kappa cDNA fragment, the MM-specific expression vector pSilencer K-IE-IgPAPE1siRNA was cloned and identified by restriction enzyme digestion And sequencing confirmed. The recombinant plasmids were transfected into KM3, HOS and MDA-231 cells by lipofection method. The specific knockout effect of APE1 on KM3 cells was analyzed by Western blot. Results The expression of APE1 protein in KM3 cells was knocked down by pSilencer K-IE-IgP-APE1 siRNA. The relative expression level of APE1 in KM3 cells transfected with siRNA was 0.118 ± 0.047, while the expression of APE1 The relative expression level was 0.988 ± 0.029, and the gene silencing efficiency was 90%. Conclusion The APE1siRNA expression vector targeting MM was successfully constructed.