慢病毒介导的小鼠CCR3基因RNAi载体的构建及其在肥大细胞中的表达

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

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CCR3mRNA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3.7×10^8 TU/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。

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 293 Tcells 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 293 Tcells and packaged with virus.Finally the high purity PDSO19-PL-CCR3 lentiviral plasmid was obtained with a virus titer of 3.7×10^8 TU/ml.The lentiviral plasmid was used to infect mouse mast cells.RT-PCR and Western Blot detection assay showed that CCR3 shRNA 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 downregulated 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.