Construction Of Fusion Gene DNA Vaccine With Gpd And Tp92 From Treponema Pallidum And Its Preliminary Study On The Immunity Responses In New Zealand White Rabbits

Objectives:(1) To construct the recombinant plasmid of Eukaryotic Expression pcDNA3.1(+)-Gpd,pcDNA3.1(+)-Tp92 and pcDNA3.1(+)-Gpd-Tp92 containing the outer membrane protein Gpd or Tp92 gene of T. pallidum and to express fusion protein in HeLa cell.(2) To observe immune response induced by nucleic acid vaccines based on outer membrane protein Gpd and Tp92 of T. pallidum in new Zealand white rabbits and evaluate its utility for DNA immunization.Methods:Primers were designed by primer 5.0 software according to the Gpd and Tp92 gene sequence of T. pallidum. Polymerase chain reaction(PCR) was used to amplify the Gpd and Tp92 gene. The fragment was directly ligated into pUCm-T vector.After identified by PCR, restriction enzyme analysis and sequence,Gpd and Tp92 gene were subcloned to pcDNA3.1(+) eukaryotic expression vector by linking reactions to construct pcDNA3.1(+)-Gpd, pcDNA3.1(+)-Tp92 and pcDNA3.1(+)-Gpd-Tp92, the recombinant plasmids were transfected into HeLa cells using liposome. After verifying that the Gpd and Tp92 antigen genes could be expressed in HeLa cells by western blotting and immunocytochemistry, three kinds of recombinant plasmids,the control plasmid pcDNA3.1(+) and PBS buffer were administered respectively in five groups of New Zealand White rabbits.Booster immunizations were employed at 2-week interval for three times.ELISA were used for the quantitative detection of the specific antibody in the sera of rabbits and the cytokine IFN-γ in rabbits spleenlymphocyte culture medium after stimulating by Gpd or Tp92. The proliferation response of spleen cells was detected by MTT assay and transformation experiment. The Gpd and Tp92 gene in muscle were identified by PCR .The significant specific antibody IgG titers were detected and the highest titer was 1:1024 in rabbits of nucleic acid vaccine groups after three times inoculations.The proliferation response of spleen cells were significantly higher than those of rabbits injected with pcDNA3.1(+)(p<0.01)All above results establish a solid basis for future studying the biological activities of Gpd and Tp92,and benefit the development of the Syphilis DNA vaccineResults:(1) The eukaryotic expression recombinant pcDNA3.1(+)-Gpd was successfully constructed, Gpd gene constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 41KDa in HeLa cells and react with positive serum from syphilis patients .(2) The eukaryotic expression recombinant pcDNA3.1(+)-Tp92 was successfully constructed, Tp92 gene constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77KDa in Hela cells and react with positive serum from syphilis patients .(3) The eukaryotic expression recombinant pcDNA3.1(+)-Gpd-Tp92 was successfully constructed, Gpd-Tp92 fusion gene constructed in pcDNA3.1(+) vector could express a fusion protein in Hela cells and react with positive serum from syphilis patients.(4) The significant specific antibody titers were detected by ELISA in three DNA vaccine groups and all of the highest titer were 1:1024 after six weeks .(5) The cytokine IFN-y in rabbits inoculated with pcDNA3.1(+)-Gpd, pcDNA3.1(+)-Tp92 and pcDNA3.1(+)-Gpd-Tp92 were increased and respectively reached 476±38.624pg/mL,483±38.884pg/mL and 543±41.625pg/mL. A significant difference was tested between the experiment groups and control group(87±13.635...