The Experimental Study On Recombinant Lentiviral Vector Vaccine Co-expressing LRG47/Rv2031c

Objective:To construct LRG47/Rv2031 c co-expressing recombinant lentiviral vector vaccine that can specifically express LRG47 and Rv2031 in macrophage, and investigated the effects and mechanisms of this vaccine on antimycobacterial activity in macrophage.Method:1. Amplifying Rv2031 c coding gene and 2031-LRG47 coding fragment by PCR,inserted into lentiviral expression vector pLenti-146-GFP, under the control of macrophage specific promoter, to obtain recombinant lentiviral vector pLenti-2031 and pLenti-2031-LRG47. These recombinant plasmids were verified by cutting with restriction enzymes and sequencing.2. The lentiviral expression plasmid( pLenti, pLenti-146-GFP, pLenti-2031 and pLenti-2031-LRG47) and lentiviral packaging plasmid( pMD2 G and pSPAX2)were co-transfected into 293 T cells according to a certain proportion respectively,collecting the corresponding lentiviral particle after 72 hours, then concentrated the virus particles through centrifugal ultrafiltration, obtaining recombinant lentivirus LV-Lenti, LV-Lenti-146-GFP, LV-Lenti-2031 and LV-Lenti-2031-LRG47. Then clearing the viral titer by counting GFP positive expression rate of cells using flow cytometry after transduced the RAW264.7 cells with lentiviral LV-Lenti-146-GFP,to confirm the optimal viral titer in the following study.3. To confirm the best time of transduction, we observed the expression of green fluorescence by fluorescence microscopy at 24 h, 48 h, 72 h and 96 h respectively after transduced the RAW264.7 cells with lentiviral LV-Lenti-146-GFP. The expression of Rv2031 and LRG47 in macrophage was detected by RT-PCR and Western-blot at the best time.4. RAW264.7 cells were transduced with above-mentioned recombinant lentivirus for 72 h, followed by infection with H37 RV or H37RV-GFP at the MOIs of5, then washed the bacteria which was not engulfed thoroughly by macrophage at 4 hpost-infection and the time is 0 h. Phagocytosis of H37RV-GFP was detected by flow cytometry at 0 h and intracellular mycobacteria survival was detected by CFU counting at the indicated time points post-infection.5. RAW264.7 cells were transduced with above-mentioned recombinant lentivirus for 72 h, then detected the following indicators to explore its possible mechanism of sterilization:(1) Detecting the expression of autophagy related protein LC3 by Western-blot and the level of autophagy of cells in each group with Cyto-ID Autophagy Detection Kit by flow cytometry.(2) Detecting the colocalization of H37RV-GFP and lysosomes under confocal microscope by immunofluorescence.(3)Detecting the expression of MHCII and CD80 on the surface of macrophage by flow cytometry.6. Immunizing the BALB/c mice with OVA, to product the OVA sensitized lymphocytes. Transducting the mouse peritoneal macrophages with above-mentioned recombinant lentivirus, then added the OVA to OVA antigen load after 48 h transduction and mixed culture with the OVA sensitized mice spleen lymphocytes after 72 h transduction. Detect the proliferation and activation of lymphocyte at the indicated time points to explore the effect of recombinant lentivirus on the antigen presentation capacity of macrophage.Results:1. Enzyme digestion and sequencing results show that the recombinant lentiviral expression plasmid pLenti-2031 and pLenti-2031-LRG47 were successfully constructed.2. Recombinant lentiviral LV-Lenti, LV-Lenti-146-GFP, LV-Lenti-2031 and LV-Lenti-2031-LRG47 were successfully packaged and the virus titers were more then 1-2 × 107 TU/ml, which have been completely conform to required transduction.The beat time of transduction is 72 h.3. RT-PCR and Western blot results show that Rv2031 and LRG47 can express in RAW264.7 cells effectively.4. Flow cytometry detection of bacterial load detection results show that the phagocytosis of H37RV-GFP by macrophages has not significantly influence after transduction with recombinant lentiviral. But compared with the control group,LV-Lenti-2031-LRG47 can significantly enhance the bactericidal capacity of Mtb by macrophages.5. Compared with the control group, LV-Lenti-2031-LRG47 can significantly improve the autophagy of macrophages and promote the colocalization of H37RV-GFP and lysosomes in macrophages.6. Compared with the control group, LV-Lenti-2031-LRG47 can significantly improve the expression of MHCII and CD80 on the surface of macrophages. At the same time, the mixed cells culture results show that LV-Lenti-2031-LRG47 can significantly enhanced the ability of macrophages to activate lymphocytes.Conclusion:1. In this research, the LRG47/Rv2031 c recombinant lentiviral expression plasmid was successfully constructed; the packaging and concentration of the recombinant lentiviral were completed, and achieved the effective expression in macrophages.2. This research confirmed that the LRG47/Rv2031 c recombinant lentiviral can enhance the bactericidal capacity of Mtb by macrophages.3. This research confirmed that the LRG47/Rv2031 c recombinant lentiviral can induce the autophagy of macrophages and promote the colocalization of H37RV-GFP and lysosomes in macrophages.4. This research confirmed that the LRG47/Rv2031 c recombinant lentiviral can improve the ability of antigen presenting in macrophages.