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

Abstract

Objective: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.Method: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.Result: 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.Conclusion: 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.

Keywords: RNA interfere; chemokine receptor 3; lentivirus; mast cell.