布鲁菌M5疫苗株感染对宿主细胞自噬溶酶体途径的影响

Effect of Brucella M5 vaccine strain infection on autophagy lysosomal pathway of host cells

通过慢病毒包装系统构建能稳定表达双荧光标记的微管相关蛋白轻链3(mCherry-GFP-LC3Ⅱ)的小鼠巨噬细胞系(RAW264.7),并探讨布鲁菌感染对自噬溶酶体途径的影响。设计并合成含mCherry-GFP-LC3Ⅱ基因的重组慢病毒表达质粒,将重组质粒与慢病毒包装质粒(pMD2.G和pspa×2.0)共同转染293T细胞,浓缩细胞上清中的病毒,用其感染RAW264.7小鼠巨噬细胞。经嘌呤霉素抗性筛选获得稳定表达mCherry-GFP-LC3Ⅱ的RAW246.7细胞株,用倒置荧光显微镜和流式细胞术分析感染效率。分别用Earle’s平衡盐缓冲液(Earle’s balanced salt solution,EBSS)和布鲁菌M5疫苗株菌株处理该细胞株,激光共聚焦显微镜下观察mCherry-GFP-LC3Ⅱ斑点。成功获得了稳定表达mCherry-GFP-LC3Ⅱ的RAW264.7细胞株,经嘌呤霉素筛选后荧光显微镜和流式细胞仪显示荧光阳性率达90%以上,EBSS和布鲁菌M5疫苗株菌株处理后自噬斑点显著增多。成功构建了能够稳定传代的mCherry-GFP-LC3Ⅱ-RAW264.7细胞株,为后续自噬相关研究建立了可靠的细胞平台。

To construct mouse RAW264.7 macrophage containing red fluorescence,green fluorescence and microtubule-associated protein light chain 3(mCherry-GFP-LC3Ⅱ),and to explore the effect of Brucella M5vaccine strain on autophagy,a recombinant lentivirus expression plasmid containing mCherry-GFP-LC3Ⅱ gene was designed and synthesized.The recombinant plasmid and lentivirus packaging plasmid(pMD2.G,pspa×2.0)were co-transfected into 293Tcells,and the supernatant containing virus was collected and concentrated to infect RAW264.7cells.The RAW246.7cell line stably expressing mCherry-GFP-LC3Ⅱwas obtained through puromycin resistance screening,and the infection efficiency was analyzed by inverted fluorescence microscope and flow cytometry.The strain was treated with Earle’s balanced salt solution(EBSS)and Brucella M5vaccine strain respectively,and the mCherry-GFP-LC3Ⅱ spots were observed under laser confocal microscope.The RAW264.7 cell line stably expressing mCherry-GFP-LC3Ⅱ was successfully obtained.After screening with puromycin,the fluorescence microscope and flow cytometry showed that the fluorescence positive rate was above 90%.After treatment with EBSS and Brucella M5vaccine strain,the autophagy spots increased significantly.In conclusion,mCherry-GFP-LC3Ⅱ-RAW264.7cell line with stable passage was successfully constructed,which established a reliable cell platform for subsequent autophagy related research.