LRG47/EBP50重组慢病毒靶向载体疫苗增强RAW264.7小鼠巨噬细胞的抗结核免疫及机制

LRG47/EBP50 recombinant lentivirus-targeted vector vaccine enhances anti-tuberculosis immunity of RAW264.7 macrophages and its mechanism

目的构建免疫相关p47 GTP酶/埃兹蛋白-根蛋白-膜突蛋白结合磷蛋白50(LRG47/EBP50)基因共表达重组慢病毒靶向载体,探讨该基因疫苗在抗结核免疫中的作用。方法利用分子克隆技术构建重组慢病毒质粒载体pLenti-EBP50-LRG47,包装成慢病毒LV-EBP50-LRG47后,H37Rv菌株感染RAW264.7小鼠巨噬细胞。荷菌量计数检测各组杀菌能力,流式细胞术检测巨噬细胞的自噬和凋亡水平,Western blot法检测诱导型一氧化氮合酶(iNOS)蛋白表达,紫外分光光度计法检测一氧化氮(NO)的表达水平。结果重组慢病毒LV-EBP50-LRG47感染巨噬细胞后,可成功过表达EBP50和LRG47,与对照组相比,LV-EBP50-LRG47可显著抑制胞内H37Rv的生长,LV-EBP50-LRG47感染巨噬细胞自噬和凋亡水平显著上调,iNOS和NO表达水平显著上调。结论LRG47/EBP50共表达的巨噬细胞自噬、凋亡增强,iNOS、NO产生增加,显著抑制胞内H37Rv的生长。

Objective To investigate the effect of the gene vaccine in anti-tuberculosis immunity by constructing immunity-related p47 GTPase/ezrin-radixin-moesin-binding phosphoprotein 50(LRG47/EBP50)gene co-expression recombinant lentivirus targeting vector.Methods Recombinant lentiviral plasmid vector pLenti-EBP50-LRG47 was established by using molecular cloning and packaged as lentivirus LV-EBP50-LRG47 and H37Rv infect macrophages.Then their bactericidal ability was tested by colony-forming units while the cellular autophagy and apoptosis was detected by flow cytometry.iNOS protein was detected by Western blotting and the expression level of nitric oxide(NO)was detected by ultraviolet spectrophotometer.Results The recombinant lentivirus LV-EBP50-LRG47 successfully up-regulated the expression of EBP50 and LRG47 after infecting macrophages.Compared with the control group,LV-EBP50-LRG47 can significantly inhibit the growth of intracellular H37Rv.The autophagy and apoptosis levels of LV-EBP50-LRG47 infected macrophages increased significantly,and the expression levels of iNOS and NO were significantly up-regulated.Conclusion LRG47/EBP50 gene co-expression enhances macrophages autophagy and apoptosis,and increases generation of iNOS and NO,which significantly inhibites the growth of intracellular H37Rv.