Autophagy Mediated Recombinant DNA Vaccine Of JE Enhances The Function Of Dendritic Cells In Balb/c Mice

Objective: To investigate the effect of autophagy mediated recombinant DNA vaccine of JE on the function of spleen dendritic cells in mice.Methods:1.Animal grouping and immunizationFifty clean healthy female Balb/c mice(4-6 weeks of age)were randomly divided into 5 groups after adaptive feeding for one week.Mice in pJME-LC3,pJME,pc DNA3.1(+),PBS group were respectively intramuscular injected with different plasmids as describled:100μg pJME-LC3 plasmid(1mg/ml,dissolved in sterile PBS)was intramuscular injected to immunize the quadriceps femoris of the left hind limb of mice for 3 times,with an interval of 2 weeks,as the experimental group,100μg of pJME plasmid(1mg/ml,dissolved in sterile PBS)was injected intramuscularly as the routine DNA vaccine immunization group,the empty vector pc DNA3.1(+)was injected intramuscularly as the negative control group,and the sterile PBS group as the blank control group.The immunization method was the same as before.The other group received intraperitoneal injection with 100μL JE live attenuated vaccine as a positive control.The used plasmids were identified by Bam H I/Eco R I double digestion and sequencing.2.Study on the effect of PJME-LC3 DNA vaccine on dendritic cell functionThree weeks after the final immunization,10 mice in each group were sacrificed and spleen were taken aseptically to separate lymphocytes and DCs.Western Blot was used to detect the immune mouse DCs in LC3 Ⅱ protein expression and immunofluorescence was used to observe the localization of pr ME-LC3 fusion protein and autophagosome in immunized mouse spleen DCs.The expression of Major histocompatibility complex Ⅱ(MHC Ⅱ),CD80 and CD86 molecules on the DCs surface was analyzed by flow cytometry.CCK8 assay was used to detect and compare the effects of DCs on the proliferation of spleen mononuclear cells in allogeneic mice.The secretion of cytokines IL-12 stimulated by dendritic cells were detected by ELISA.Results:1.pJME-LC3 fusion protein co-locates with autophagy in immunized mouse spleen DCsLC3 Ⅱ protein expression increased in the DCs of pJME-LC3 group,prompt autophagy activity in DCs.The results of confocal microscopy showed that the expression of DCs intracytoplasmic fusion protein in pJME-LC3 group was significantly higher than that in the conventional JE DNA vaccine control group.At the same time,it was observed that the autophagosomes in the DCs of the pJME-LC3 group were co-localized with the fusion protein,thus presenting yellow color,while the DCs in the pJME group did not fuse with each other.2.pJME-LC3 promotes the expression of MHC Ⅱ and CD80,CD86 molecules.Flow cytometry was used to detect the expression of marker molecules on the surface of DCs in different immunized groups,and the proportion of positive cells was compared.The expression of MHC Ⅱ and CD80 molecules of DCs in pJME-LC3 experimental group was significantly higher than other groups.The expression of CD86 molecules of DCs in pJME-LC3 experimental groups was significantly higher than pJME group,but there was no statistical difference between PJME-LC3 group and JEV-L group.3.pJME-LC3 promotes the secretion of DCS Th1 cytokines in spleen DCsThe cytokine IL-12(55.35±11.51)pg/m L was significantly higher in pJME(37.98±7.59)pg/m L and JEV-L(36.46±6.64)pg/m L(**P<0.01).There was no significant difference between the PJME group and the JEV-L group.4.pJME-LC3 enhanced the stimulating effect of spleen DCs on lymphocyte proliferationThe stimulation index of DCs in pJME-LC3 group(6.09 ± 0.20)was significantly higher than that of pJME group(5.06± 0.31)(*P<0.05)and JEV-L immunization group(5.17 ± 0.23)(**P<0.01).There was no significant difference between the PJME group(5.06 ± 0.31)and the JEV-L group(5.17 ± 0.23).Conclusion:1.pJME-LC3 can promote the expression of plasmid encoded protein in DCs,and improve the antigen presentation ability of spleen DCs by enhancing the autophagy activity of spleen DCs in immunized mice.2.pJME-LC3 can enhance the ability of DCS to stimulate the proliferation of spleen lymphocytes,and increase the secretion of Th1 type cytokine(IL-12)in DCs,so as to promot the cellular immune effect of pJME-LC3.