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目的:构建并鉴定靶向人神经菌毛素-1(NRP-1)基因的小发卡RNA的慢病毒载体。方法:针对NRP-1mRNA设计了4条shRNA,并构建pGCSIL-RFP-shNRP1慢病毒质粒,PCR扩增阳性克隆并测序鉴定。用pGCSIL-RFP-shNRP1、pHelper1.0和pHelper2.0质粒共转染293T细胞包装产生慢病毒,测定病毒滴度。将慢病毒干扰RNA及含有NRP-1过表达载体共转染293T细胞,Western blot法检测Flag表达,观察NRP-1蛋白相对表达抑制效果。结果:PCR和测序结果与设计的干扰序列一致,病毒滴度达1×109Tu/mL。转染细胞中NRP-1蛋白相对表达显著降低。结论:成功构建了高效率的人NRP-1基因shRNA慢病毒载体。
OBJECTIVE: To construct and identify a lentiviral vector targeting small hairpin RNA of human neuropilin-1 (NRP-1) gene. Methods: Four shRNAs were designed for NRP-1 mRNA and a pGCSIL-RFP-shNRP1 lentiviral plasmid was constructed. The positive clones were amplified by PCR and sequenced. 293T cells were co-transfected with pGCSIL-RFP-shNRP1, pHelper1.0 and pHelper2.0 plasmids to produce lentivirus and the virus titer was determined. 293T cells were co-transfected with lentiviral RNA and NRP-1 overexpression vector, and the expression of Flag was detected by Western blot. The inhibitory effect of NRP-1 protein expression was observed. Results: PCR and sequencing results were consistent with the designed interference sequences. The virus titer reached 1 × 109Tu / mL. The relative expression of NRP-1 protein in transfected cells was significantly reduced. Conclusion: A highly efficient human lentiviral vector encoding human NRP-1 gene was successfully constructed.