Construction Of A Therapeutic DNA Vaccine Enhancing Autophage And Delivering Mycobacterium Tuberculosis Persistent Antigen To Specific Target

Objective:To construct a therapeutic DNA vaccine which can targetedly express the IRGM1protein and encode Mycobacterium tuberculosis(Mtb) persistent antigen in macrophage. To explore the effect and mechanism of the therapeutic DNA vaccine in battling against MTB from the molecular, cellular and animal level. It is expected to overcome latent infection, multidrug resistance and reinfection problem of Mtb therapy.Methods:1. The sequence of IRGM1and Rvl733c/Rv2031c/Rv2626c were amplif ied by PCR and then inserted into pET-32a(+) plasmid.The promoter CMV of the eukaryotic coexpression plasmid pBudCE4.1was replaced by Macrophage-s pecific promoter SP107to form pBudSP. All the recombinant plasmids above were sequenced respectively. The sequence of IRGM1was subcloned into pBu dSP at Sal Ⅰ/BamH Ⅰ site, downstream of the promoter SP107, to form pBudS P-L.Whereas Rv1733c/Rv2031c/Rv2626c were subcloned into pBudSP-L at N ot Ⅰ/Kpn I site, downstream of the promoter EF-la, to form recombinant plasm ids pBudSP-L-1733c/2031c/2626c.2. The sequence of RFP and IRGM1without termination codon were amp lified from plasmid pERLO by PCR, then inserted into plasmid pCDNA3.1(+) and pET-32a(+) to form pCDNA3.1(+)-RFP and pET32a(+)-IRGM1②,respective ly. All the recombinant plasmids above were sequenced. After that, the sequen ce of RFP was subcloned into the recombinant plasmid pBudSP to form the p lasmid pBudSP-RFP, and then the sequence of IRGM1without termination cod on was subcloned into pBudSP-RFP at Sal Ⅰ/BamH I site to form pBudSP-LR. At last, The sequences of Rv1733c/Rv2031c/Rv2626c were subcloned into pBudSP-LR at Not Ⅰ/Kpn I site to form Macrophage-specific eukaryotic coexp ression plasmids pBudSP-LR-1733c/2031c/2626c. Plasmids pBudSP-LR-1733c/2031c/2626c were transfected into RAW264.7cells (a cell line of murine m acrophage), respectively. The expression of IRGM1and Rv1733c/Rv2031c/Rv2626c gene were detected by fluorescence.3. The sequence of Rv1733c without termination codon was amplified by PCR, and then inserted into plasmid pCDNA3.1(-) to form pCDNA3.1(-)-1733②. At the same time, the sequence of eGFP was amplified from plasmid pEG FP-N1by PCR, and inserted into plasmid pET-32a(+) to form pET32a(+)-GFP. All the plasmids above were sequenced. After that, the sequence of eGFP was subcloned into pCDNA3.1(-)-1733②at BamH Ⅰ/Kpn I site to form the plasmi d pCDNA3.1(-)-1733G, then the sequence of1733c-GFP was subcloned into p BudSP-LR at Not Ⅰ/Kpn Ⅰ site to form Macrophage-specific eukaryotic coexpr ession plasmid pBudSP-LR-1733G. The plasmid pBudSP-LR-1733G was transfe cted into RAW264.7cells by electroporation. The expression of IRGM1and R v1733c gene were detected by fluorescence and RQ-PCR.4. The sequence of RFP was subcloned into the plasmid pBudCE4.1at HindⅢ/Xba I site to form control plasmid pBudCE-RFP. The plasmid pBudSP-RFP, pBudSP-LR-1733G, pBudCE-RFP and pERLO were transfected into RAW264.7,293T, Hela and HepG2cells, respectively. The specificity of SP107was detected by fluorescence in different groups.Results:1. The eukaryotic coexpression plasmids pBudSP-L-1733c/2031c/2626c c arrying the sequences of Macrophage-specific promoter SP107, murine IRGM1and Mtb persistent antigens Rv1733c/Rv2031c/Rv2626c were successfully con structed.2. The eukaryotic coexpression plasmid pBudSP-LR-1733c/2031c/2626c c arrying the coding sequences of murine IRGM1-RFP and Mtb persistent antige ns Rvl733c/Rv2031c/Rv2626c were successfully constructed. The recombinan t plasmids pBudSP-LR-1733c/2031c/2626c were successfully transfected into RAW264.7, and the expression of IRGM1was detected by red fluorescence, s uccessfully.3. The eukaryotic coexpression plasmid pBudSP-LR-1733G carrying the co ding sequences of murine IRGM1-RFP and MTB persistent antigen1733c-GFP was successfully constructed. The recombinant plasmid was successfully transf ected into RAW264.7. The expression of target genes were detected by fluores cence and RQ-PCR, successfully.4. The control plasmid pBudCE-RFP was successfully constructed. The RF P of plasmid pBudSP-RFP and pBudSP-LR-1733G can only be observed in R AW264.7, whereas the RFP of plasmid pBudCE-RFP and pERLO can be obser ved in RAW264.7,293T, Hela and HepG2cells.Conclusion:1. Murine IRGM1and MTB persistent antigens Rv1733c/Rv2031c/Rv2626c can be coexpressed in macrophage.2. IRGM1can express fused with RFP and Rv1733c can express fused w ith eGFP, which can make observation for the expression of IRGM1and Rv1733c in Macrophage or animal experiments more conveniently.3. SP107has high specificity, which suggested the macrophage-specific ex pression of DNA vaccine.