The Study Of The Impacts And Mechanisms Of Lentiviral Vector-mediated EBP50 Over-expression On The Antimycobacterial Activity Of Macrophage

Objective:To construct and pack a recombinant lentiviral vector expression EBP50,investigate the impacts of EBP50-overexpression on the antimycobacterial activity of macrophage,and probe the related mechanisms by analyzing the colocalization rate of Mtb containing phagosome and lysosome,detecting the expression of iNOS,the production of NO,the autophagy and apotosis level of macrophages.1.The construction of recombinant lentiviral vector:The coding sequence of EBP50 were amplified from the pLEM plasmid by RT-PCR,then insered into lentiviral expression vector pLenti-146-GFP plasmid,constructing recombinant lentiviral vector pLenti-146-EBP50 plasmid.It was identified by using restriction enzymes and then sequenced.2.Lentivirus Packaging and calculating the viral titer:The lentiviral vector plasmid(pLenti-146-EBP50?pLenti-146-GFP and pLenti)and lentiviral packaging plasmid(pMD2.G and psPAX2)were cotransfected into 293T cells respectively,collecting the supernatant lentiviral particle after 72 hours,then centrifugal ultrafiltration to concentrate virus particles,To obtain recombinant lentivirus LV-EBP50?LV-Lenti-GFP and LV-Lenti.The RAW264.7 cells were transduced with lentiviral LV-Lenti-GFP,then to clear the viral titer by counting GFP positive expression rate of cells using FCM(flow cytometry),confirming the optimal viral titer finally.3.The identification of the recombinant lentiviral' s expression effect:To confirm the best time of transduction,we observed the expression of fluorescence by fluorescence microscopy at 24h?48h and 72h after transducing into RAW264.7 in MOI=10,The expression of EBP50 on macrophage was detected by RT-PCR and Western blot after the best time.4.To explore the impacts of EBP50-overexpression on the antimycobacterial activity of macrophage:The RAW264.7 cells were infected by H37Rv after72 hours' recombinant Lentivirus EBP50 transduction in MOI=10.Then cultivate and count the Mtb colonies after lysesing cells at different time point.5.To explore the mechanisms of EBP50-overexpression on the antimycobacterial activity of macrophage:The RAW264.7 cells were infected by H37Rv after 72 hours'recombinant Lentivirus EBP50 transduction in MOI=10:(1)The impacts of EBP50-overexpression on the expression and localization of iNOS:iNOS was detected by Western-blot;To detect the colocalization rate of phagosome and INOS on macrophage by immunofluoresent assay after 24 hours'transduction;(2)The impacts of EBP50-overexpression on the production of NO:The content of NO was detected by nitrate reductase method;(3)The impacts of EBP50-overexpression on the maturation of phagosome:To detect the colocalization rate of phagosome and lysosome on macrophage by immunofluoresent assay after 24 hours' transduction;(4)EBP50-overexpression on the autophagy of macrophage:the autophagy on macrophage were detected by FCM(flow cytometry)by Cyto-ID Autophagy detection kit;LC3 was detected by Western-blot;(5)the apoptosis on macrophage were detected by FCM(flow cytometry)by Annexin V-EGFP.Results:1.recombinant lentiviral vector pLenti-146-EBP50 is successfully constructed.2.recombinant lentivirus LV-EBP50?LV-Lenti-GFP? LV-Lenti were obtained,and viral titer are above 1.0×107 TU/ml.The recombinant lentivirus can be used for cellular level's research.At the same time,It is found that The fluorescence expression was strongest after 72 hours' transduction and cell state is good.3.EBP50 successfully detected by RT-PCR and Western blot from transduced RAW264.7 cells.4.recombinant Lentivirus LV-EBP50 on macrophage antimycobacterial activity is stronger than LV-Lenti-GFP.5.The level of macrophage inducible nitric oxide Synthase and NO production is higher than LV-Lenti-GFP,at the same time,The recombinant Lentivirus LV-EBP50 can not promote the colocalization rate of phagosome and INOS.The level of macrophage autophagy and LC3 expression of LV-EBP50 is higher than LV-Lenti-GFP,at the same time,The recombinant Lentivirus LV-EBP50 can promote the colocalization rate of phagosome and lysosome.The apoptosis level of LV-EBP50 is higher than LV-Lenti-GFP.Conclusion:1.recombinant lentiviral vector pLenti-146-EBP50 is successfully constructed,completed lentivirus packaging and can effective express on RAW264.7 cells.2.The EBP50 gene show macrophage antimycobacterial activity on is higher in vitro.3.recombinant Lentivirus EBP50 on macrophage antimycobacterial activity can improve from two aspects:(1)promote the colocalization rate of phagosome and lysosome by the autophagy-lysosomal pathway.(2)promote the level of apoptosis.