Immuno-regulation Of T-bet Adjuvant On Ag85B DNA Vaccination Against Tuberculosis

ObjectivesTo construct the eukaryotic expression plasmids of pcDNA3.1(-)-T-bet and pcDNA3.1(-)-Ag85B. Next, to investigate the new Ag85B DNA complex vaccine based on the genetic adjuvant of T-bet regulating immuno response via experiments in vitro and in vivo, which provides new theoretical and experimental evidences for the prevention and treatment of Thl imbalance disease including tuberculosis.Methods1. According to the full-length sequence of the target gene Ag85B and T-bet in GenBank and restriction sites of pcDNA3.1(-)、pET-28a(+) plasmids, the specific primers were designed using the softwares of DNAClub and Premier Primer5. Ag85B and T-bet gene were amplified with specific primers with PCR or RT-PCR, the corresponding PCR products and pcDNA3.1(-) plasmid were subjected to double restrictions with BamH Ⅰ/Hind Ⅲ and Xho Ⅰ/EcoR I, respectively. Then, purified the DNA fragments and constructed pcDNA3.1(-)-Ag85B and pcDNA3.1(-)-T-bet plasmids using T4DNA ligase, the recombinant plasmids were identified by PCR, and digested by restriction endonucleases and sequencing.2. In addition, Ag85B fragment double digested from the recombinant plasmid pcDNA3.1(-)-Ag85B was subcloned into the eukaryotic expression plasmid pET-28a(+). Then, recover the target gene and the vector fragment, ligate them using T4DNA ligase and transform into E.coli DH5a to screen the recombinant plasmid. Finally, the recombinant plasmid pET-28a(+)-Ag85B was identified by PCR and digestion with restriction enzymes.3. The recombinant plasmid pET-28a(+)-Ag85B obtained above was transformed into E.coli BL21(DE3) to express rAg85B by the induction of IPTG. The expressed product was analyzed using SDS-PAGE and purified by Ni-IDA His-band resin.4. The recombinant plasmids pcDNA3.1(-)-Ag85B and pcDNA3.1(-)-T-bet were transfected into RAW264.7cells using lipofectamineTM2000reagent. Then the cells were incubated in a5%CO2incubator at37℃. After24or48hours of transfection, the expression of Ag85B and T-bet protein were detected by Western blotting.5. BALB/c mice were immunized by three intramuscular inoculations with pcDNA3.1(-)-T-bet in combination with pcDNA3.1(-)-Ag85B. Two weeks after the last immunization, the anti-Ag85B antibody titres in sera were tested by ELISA. Meanwhile, the mouse spleen lymphocytes were cultured in the context of Ag85B, and then tested the secretion of cytokines in culture fluid by ELISA.Results1. The PCR products of the target gene amplified from genomic DNA of M.tuberculosis standard strain H37Rv and total RNA of normal mouse spleen lymphocytes were identified to be about1091bp and1608bp using DNA gel electrophoresis, respectively.2. PCR, double digestion with restriction endonucleases and sequencing comfirmed that the recombinant plasmids pcDNA3.1(-)-Ag85B, pcDNA3.1(-)-T-bet, pET-28a(+)-Ag85B were constructed successfully.3. After induction of IPTG, the recombinant protein rAg85B was expressed in E.coli BL21(DE3) which transformed with the pET-28a(+)-Ag85B plasmid, the molecular weight was as same as the predicted value. Subsequently, the protein was collected by affinity chromatography.4. Different concentrations of eukaryotic expression plasmid pcDNA3.1-Ag85B, pcDNA3.1-T-bet were transfected to RAW264.7cells. Western blotting comfirmed FLAG-Ag85B and FLAG-T-bet protein were expressed successfully in transfected cells and positively correlated with the recombinant plasmid dose.5. ELISA results showed that the anti-Ag85B antiboby titre was no significant difference between vector-and T-bet-group. In comparision with Ag85B group, IgG2a titer was significantly increased in Ag85B/T-bet group, while IgGl level was obviously decreased.6. The levels of IFN-γ/IL-2/IL-4/IL-10were no significant difference between T-bet group and Ag85B/T-bet group. While compared to Ag85B, Ag85B/T-bet complex induced obviously higher IFN-γ and IL-2levels with the lower of IL-4and IL-10.ConclusionsThe experiments preliminarily clarified that T-bet enhances Ag85B-specific antibody responses, which is dominance of IgG2a in sera. In addition, it stimulates the secretion of cytokines and converts T cell subsets to a Thl-predominant immune response. It lays a good foundation for further study of its anti-TB effect, as well as provides a theoretical basis and new ideas for in depth understanding of the meaning and application prospects of the immune-enhancing adjuvants.