基于慢病毒系统的双荧光标记多功能自噬流监测系统建立与应用

目的:构建能够用于稳定动态监测细胞自噬流变化和过表达基因的红色荧光蛋白-绿色荧光蛋白-鼠源LC3融合慢病毒多功能表达载体(PCDH-Duo-mRFP-eGFP_(ph)-LC3_(rat),PCDH-Duo),并构建小鼠腹腔巨噬细胞Raw264. 7稳转株观察自噬流变化。方法:应用基于PCR精确合成mRFP-eGFP_(ph)-LC3_(rat)融合全基因,将其克隆至慢病毒表达载体PCDH-CMV-MCS-EF1a-GFP中,重组质粒经菌落PCR、酶切及测序分析正确无误后,包装慢病毒,转染Raw264. 7细胞,并利用流式分选术获取稳转株,经氯喹抑制自噬模型及Western blot鉴定eGFP蛋白表达确认其可靠性。结果:成功构建了PCDH-Duo重组慢病毒质粒,包被慢病毒并获得Raw264. 7稳定细胞系(Raw264. 7-PCDHDuo),可稳定表达双荧光蛋白,经3mmol/L氯喹作用6h后,能够稳定准确指示自噬流变化。结论:成功构建了基于慢病毒系统的双荧光标记多功能自噬流监测系统,为研究细胞自噬与编码基因及非编码基因之间的关系提供了方便有力的工具。

Establishment and Application of Dual Fluorescent Labeling Multi-functional Autophagy Flux Monitoring System Based on Lentiviral System

Objective: To construct a red fluorescent protein-green fluorescent protein-murine LC3 fusion multi-lentiviral expression vector (PCDH-Duo-mRFP-eGFP_ph-LC3_rat, PCDH-Duo),which can be used to stably monitor the changes of autophagy flux and overexpression genes. Changes in autophagic flow were observed in the mouse peritoneal macrophage Raw264.7 stable strain.Methods:The mRFP-eGFP_ph-LC3_rat fusion gene was synthesized by PAS and cloned into the lentiviral expression vector PCDH-CMV-MCS-EF1a-copGFP. After the recombinant plasmid was correctly analyzed by PCR, enzyme digestion and sequencing, the lentivirus was packaged. Raw264.7 cells were transfected, and stable cells were obtained by FACS. The reliability of the eGFP protein expression system was confirmed by CQ autophagy inhibition model and Western blot.Results:The recombinant plasmid of PCDH-Duo lentivirus was successfully constructed, which was coated with lentivirus and obtained stable cell line of Raw264.7. The expression of double fluorescent protein was stable. After induction by 3mmol/L CQ for 6h, it was stable and accurate. The phasing changes.Conclusion:The dual-fluorescence multi-function autophagic flux monitoring system based on lentivirus system was successfully constructed, which provides a convenient and powerful tool for studying the relationship between autophagy and coding genes and non-coding genes.