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

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.目的：构建筛选靶向趋化因子受体3（CCR3）-RNA干扰慢病毒表达载体，感染小鼠肥大细胞，观察该基因在肥大细胞中的表达及该病毒载体的干扰效率，为研究变应性鼻炎的发病机制奠定基础。 方法：首先构建3对CCR3-短发夹RNA（CCR3-shRNA）序列，然后在shRNA慢病毒载体中分别插入3对双链的shRNA oligo，3个shRNA慢病毒重组质粒构建成功后，将重组载体和病毒包装的辅助质粒共转染293T细胞，从而获得慢病毒质粒，并将其转染入体外培养及纯化小鼠骨髓源性的肥大细胞。qRT-PCR检测肥大细胞CCR3 mRNA的表达，Western Blot检测肥大细胞CCR3蛋白的表达。 结果：经测序证实，成功构建CCR3-shRNA的慢病毒载体，转染293T细胞并进行病毒包装，最终获取高纯度的PDSO19-PL-CCR3慢病毒质粒，病毒滴度为3.7×10⁸TU/ml。以该慢病毒质粒感染小鼠肥大细胞，qRT-PCR和Western Blot检测示：CCR3-shRNA降低了小鼠肥大细胞的CCR3基因在mRNA和蛋白水平的表达。 结论：成功构建了CCR3基因RNAi慢病毒表达载体质粒，发现其下调了小鼠肥大细胞CCR3基因mRNA及蛋白水平的表达，为进一步研究其在变应性鼻炎发病机制中的作用打下了基础。.