Construction and effect of Neuropilins-2 eukaryotic expression vector for RNA interference / 第三军医大学学报

ABSTRACT

Objective To construct Neuropilins-2 eukaryotic expression vector for RNA interference.Methods Recombinant targeting on gene NRP2 was designed and established with plasmid pGenSil-1 based on NRP2 cDNA equences of Genomes.Two pairs of oligonucleotides were synthesized according to the Tuschl and inserted into plasmid pGenSil-l to generate siRNA eukaryotic expression vector,DH5? strains were transformed,plasmid were extracted,and recombinant vectors were identified by the restriction map and the sequence analysis.The recombinant plasmid(pGenSil-NRP2) was transfected into the cultured LOVO cells.At 48 h after transfection,the whole cell protein was extracted,and the protein level was detected by Western blotting with mouse-anti-human NRP2 monoclonal antibody.Results Recombinant plasmids were completely coincided with the designs by the restriction map and the sequence analysis.pGenSil-NRP2 expression vector into LOVO cells down-regulated the protein level of NRP2 at 48 h after transfection.The recombinant eukaryotic expression vector were constructed successfully.Conclusion siRNA recombinant can be constructed successfully by RNAi technique for inhibiting NRP2 expression.