Construction Of A Recombinant Mycobacterium Smegmatis Therapeutic Vaccine Delivering EBP50/LRG47 To Specific Target

Objective:To construct a recombinant Mycobacterium smegmatis strain that can targetedly deliver the eukaryotic coexpression plasmid encoding mouse EBP50 and LRG47 into macrophage. To explore the effect and mechanism of the recombinantMycobacterium smegmatis strain in battling against Mycobacterium tuberculosis(MTB) from the molecular and cellular level. It is expected to overcome latent infections^ multiple-drug resistance and reburning problem of MTB therapy. We hope it can lay a theoretical foundation for opening a new thinking of MTB control.Methods:1. The coding sequence of EBP50 and LRG47 were amplified from the total RNA of RAW264.7 cells (a cell line of murine macrophage) by RT-PCR, inserted into pCDNA3.1 plasmid and then sequenced respectively. The coding sequences of EBP50,LRG47 and OriM was subcloned into the eukaryotic coexpression plasmid pBudCE4.1 by HindⅢ/BamHl, KpnⅠ/XhoⅠand NheⅠMCS sites respectively to form the plasmid pLEM. And then the plasmid pLEM was transfected into 293T cells (a cell line of human embryo kidney). The expression of these genes was detected by RT-PCR, Westernblot.2. The coding sequence of EBP50 and RFP were amplified from the plasmid pLEM and pSIREN-DNA-DsRed-Express respectively by PCR.EBP50 was inserted into the eukaryotic coexpression plasmid pCDNA3.1 by HindlⅢ/EcoRⅠMCS sites to form the plasmid pCDNA3.1-EBP50. RFP was inserted into the eukaryotic coexpression plasmid pCDNA3.1 by EcoRllXbal MCS sites to form the plasmid pCDNA3.1-RFP. After sequencing, The coding sequences of EBP50 was subcloned into the recombinant plasmid pCDNA3.1-RFP by HindⅢ/EcoRl MCS sits to form the plasmid pCDNA3.1-EBP50-RFP.And then the coding sequences of EBP50-RFP,LRG47 and Orim was subcloned into the eukaryotic coexpression plasmid pBudCE4.1 by HindⅢ/BamHl, KpnⅠ/XhoⅠand Nhel MCS sites respectively to form the plasmid pERLO. The plasmid pERLO was transfected into 293T cells (a cell line of human embryo kidney). The expression of these genes was detected by RT-PCR,fluorescence and Westernblot.3. The effects of Msl-2c on the endogenous expression of EBP50 and LRG47 were investigated by the infection (PDMs) with Msl-2c. The recombinant plasmids pLEM and pERLO were electroporated into Msl-2c to form recombinant Mycobacterium smegmatis rMS-pLEM and rMS-pERLO.The targeted property of the recombinant Mycobacterium smegmatis was demonstrated by acid-fast staining and fluorescence in RAW264.7 and peritoneum-derived macrophages(PDMs).Results:1. The eukaryotic coexpression plasmid pLEM carrying the coding sequences of murine EBP50, murine LRG47 and OriM was successfully constructed. The expression of EBP50 and LRG47 in pLEM transfected 293T cells was successfully detected by RT-PCR,Westernblot.2. The eukaryotic coexpression plasmid pERLO carrying the coding sequences of murine EBP50-RFP, murine LRG47 and OriM was successfully constructed. The expression of EBP50 and LRG47 in pERLO transfected 293T cells was successfully detected by RT-PCR,fluorescence and Westernblot. The fusion expression of EBP50 and RFP in pERLO transfected RAW264.7cells was successfully detected by fluorescence.3. Msl-2c can up-regulate the endogenous expression of EBP50 and LRG47 in PDMs. The recombinant M.smegmatis rMS-pLEM and rMS-pERLO which can target to Macrophage were successfully constructed.Conclusion:1. Murine EBP50 and Murine LRG47 can be coexpressed in eukaryotic cell.2. EBP50 can expresse fusingly with RFP, which can observate the expression of EBP50 in Macrophage in animal experiments easily. 3. RMs carrying plasimd pLEM and pERLO can target to Macrophage4. Msl-2c can up-regulate the expression of EBP50 and LRG47 in PDMs.