[Construction of mouse CCR3 gene RNAi lentivirus vector and its expression on mast cells].

The aim of this study is to screen the targeting chemokine receptor 3-RNA interference (CCR3-RNAi) lentiviral expression vector, infect mouse mast cells,observe the expression of this gene in mast cells and the interference efficiency of the virus vector.The pathogenesis of allergic rhinitis lays the foundation.Three pairs of CCR3-shRNA sequences were constructed,and three pairs of double-stranded shRNA oligo were inserted into shRNA lentiviral vectors to construct three shRNA lentiviral recombinant plasmids.The recombinant vector and virus-packed auxiliary plasmids were co-transfected into 293T cells to obtain lentiviral plasmids.The lentiviral plasmids were then transfected into mouse bone marrow-derived mast cells in vitro and purified. The expression level of CCR3 mRNA in mast cells was verified by qRT-PCR,and the expression level of CCR3 protein in mast cells was detected by Western Blot. It was confirmed by sequencing that the lentiviral vector of CCR3 shRNA was successfully constructed, transfected into 293T cells and packaged with virus. Finally the high purity PDSO19-PL-CCR3 lentiviral plasmid was obtained with a virus titer of 3.7×10⁸TU/ml.The lentiviral plasmid was used to infect mouse mast cells.RT-PCR and Western Blot detection assay showed that CCR3shRNA reduced the expression of CCR3 gene in mouse mast cells at the level of mRNA and protein. The CCR3 gene RNAi lentivirus expression vector was successfully constructed.It was found that it downregulated the expression level of CCR3 gene mRNA and protein in mouse mast cells,which laid the foundation for further research on its role in the pathogenesis of allergic rhinitis.