Aim: To construct and identify a recombinant lentiviral vector containing shRNA for human neuropilin-1 (NRP-1) gene.
Methods: Four shRNA targeting the NRP-1 mRNA were designed to construct the pGCSIL-RFP-shNRP1 lentivirus vectors. The positive clone was chosen and confirmed by PCR and DNA sequencing. 293T cells were cotransfected with pGCSIL-RFP-shNRP1, pHelper1.0 and pHelper 2.0 to package the lentivirus and the titer of the virus was tested. After lentivirus-shRNA and over-expression plasmid containing NRP-1 were transfected into 293T cells, Western blotting was used to determine the expression of Flag gene in order to observe the inhibited efficacy of relative NRP-1 expression.
Results: PCR analysis and DNA sequencing demonstrated that the shRNA sequence was consistent with the human NRP-1. The titer of the recombinant lentiviral vector was 1×10(9); Tu/mL. The relative expression of NRP-1 protein in the transfected cells significantly decreased after treated with lentiviral-shRNA.
Conclusion: We have constructed successfully the effective recombinant lentiviral vector containing shRNA for human NRP-1 gene.