Construction Of Fusion Gene DNA Vaccine With MOMP From Chlamydophila Pneumoniae And Its Preliminary Study On The Immunity Response In BALB/C Mouse

Objectives:This study was disigned to construct Chlamydophila pneumoniae (Cpn) major outer membrane protein (MOMP) monogenic nucleic acid vaccine, MOMP-IL-2 fused gene vaccine and animal model. The immune effect of the monogenic and the fused gene vaccine was compared to provide theoretical and experimental evidence for the development of high performance and new-style Cpn nucleic acid vaccine.Methods:The gene sequence of Cpn MOMP and human IL-2 was analyzed by Primer software to design specific primer . The total MOMP and human IL-2 genes were amplified by PCR and RT- PCR , the amplicon was linked by recombination PCR. The PCR product was digested by restriction enzyme, and then cloned into the pcDNA3.1(+) eukaryotic expression vector; After was verified by double enzyme digestion and sequencing. The positive expression vectors were large-scale prepared for nucleinic acid vaccine use. 100μg of each pcDNA3.1(+)-MOMP,pcDNA3.1(+)-IL-2,pcDNA3.1(+)-MOMP-IL-2 and empty vector pcDNA3.1(+) were injected into the BALB/c mouse's quadriceps femoris every time. Two weeks after the last injection, Cpn MOMP expression in mouse tissue was detected by immunohistochemical method. Mouse spleen cells??e isolated at sterile condition, the IFN-γlevel in the culture supernatant was detected by ELISA (double antibody sandwich method) after stimulated with specific recombination proteins. Specific antibody level of MOMP and MOMP-IL-2 in BALB/c mouse serum was detected by indirect ELISA. Specific proliferative response of plenic lymphocytes was assessed by MTT and lymphocyte transformation test. Results:1.This study have successfully constructed the MOMP,IL-2 and MOMP-IL-2 fusion gene prokaryotic and eukaryotic expression vector.2. This study also have successfully constructed the pcDNA3.1(+)-MOMP monogenic expression vector and the pcDNA3.1(+)-MOMP-IL-2 fused vector. and these recombinant plasmids could transcript and express effectively.3.Specific antibody generated after inoculated by pcDNA3.1(+)-MOMP monogenic nucleic acid vaccine, the titer reached the highest 1:1280 after 6 weeks' inoculation.4. In the pcDNA3.1(+)-MOMP monogenic nucleic acid vaccine and pcDNA3.1(+)-MOMP-IL-2 fused gene vaccine groups, the IFN-γreached 586±42.3pg/mL and 695±43.7pg/mL, respectively.5. After stimulated by corresponding antigen, the stimulation index of pcDNA3.1(+)-MOMP group and pcDNA3.1(+)-MOMP-IL-2 group were 1.508±0.010 and 1.573±0.012, respectively, which was higher than control group (1.374±0.006,1.254±0.004).6 Cpn MOMP gene exist in mouse muscle cell.Conclusion:1.Strong cellar immunity and humoral immunity can be induced by nucleic acid vaccines of pcDNA3.1(+)-MOMP and pcDNA3.1(+)-MOMP-IL-2 in BALB/c mouse.2. There are not obviously difference between the efficien??f specific antibody induced in double gene nucleinic acid vaccine goups and monogenic nucleinic acid vaccine groups,and the former could induce better powerful and lasting cellullar immunologic response...