Construction Of Myelin Proteolipid Protein Gene Vaccine And Preliminary Characterization Of Its Immune Effects

Myelin proteolipid protein（PLP）is the most abundant protein of the myelin in the central nervous system,which is represented in the brain,spinal cord and thymus.It plays a major role in maintaining the structural and functional integrity of myelin,promoting the maturation of oligodendrocytes,Singnal transduction between oligodendrocytes and axons.Animal studies have shown that the models of experimental allergic encephalitis（EAE）,as well as relapse remitting EAE（RR-EAE）that similar to multiple sclerosis（MS）induced by PLP peptides(e.g.PLP_139-151、PLP_178-191、PLP_180-199).The animal model of EAE induced by PLP had the characteristics of high success rate and stablility,which was considered a better antigen of EAE animal model.The PLP coding sequence was amplified from the RT-PCR of BALB/c mice brain tissue,and cloned into the eukaryotic expression vector pVAX1 for constructing the DNA vaccine pVAX1-PLP.DNA vaccine pVAX1-PLP was identified by bacteria-liquid PCR,restrictive digestion analysis of recombinant plasmid and sequencing.Forty-two BALB/c mice with 6-8 weeks old selected and randomly divided into 3 group.Recombinant plasmid group were injected with pVAX1-PLP by muscular injection（i.m）with 18 and control groups with pVAX1 or saline with 12 in each.The behavioral changes of mice were observed,and the blood and brain tissue were collected regularly.Whole blood were collected for T cell subpopulation analysis by flow cytometry.Serum from immunized animals were examined for total Ig G and IFN-γby ELISA.Mouse brain tissues were obtained to Hematoxylin-Eosin staining and Luxol Fast Blue staining.Finally,the obtained serum were used for immunofluorescence staining.It is showed that anti-PLP were verified after 8 week and abnormal cytokine was detected after 10 week in female mice,compare with not significant in male mice.Immunized mice was scored between 1-3 points by Weaver’s 15-point.It was observed that blood vessel cuffs and a few inflammatory cell infiltrate in HE staining,and the demyelinating in brain white matter nerve was found in Luxol Fast Blue staining in both male and female mice.These results indicated that the female mice developed an immune response.In this study,BALB/c mice were immunized with gene vaccineand and the EAE mouse model was initially constructed for the first time to build a strong foundation of the discussion of the EAE autoimmunity mechanism...