| In this study, CpG-ODN were synthesized and cloned into downstream of pcDNA-PCV2-ORF2, constructed a novel DNA vaccine pcDNA-PCV-ORF2-CpG. In order to study on the adjuvants immunocity, five kinds of pig interleukin were selected, including IL-2, IL-4, IL-6, IL-6/4 and IFN-γ. Piglets were immunized with pcDNA-PCV2-ORF2, pcDNA-ORF2-CpG and 5 kinds of intefleukins by the routes of intramuscular injection. Indirect ELISA and Fluorescence Activated Cell Sorter (FACS) methods were employed respectively to detect humoral and cellular immune responses.To produce the DNA vaccine, the scaled extract of plasmid was employed and the DNA vaccines were entrapped with chitosan-nanoparticle (CNP). Piglets were immunized with 250ug,500ug of pcDNA-PCV2-ORF2 and pcDNA-ORF2-CpG DNA vaccine, and by the proportion of 1: 1 respectively by the route of intramuscular co-injection of IL-2, IL-4, IL-6, IL-6/4, IFN-γ. Indirect ELISA methods were employed to detect the anti-PCV2 antibody and Fluorescence Activated Cell Sorter (FACS) was used to detect the number of CD3+, CD4+ and CD8+ T lymphocytes.The results showed the number of CD~3+ T-lymphocytes and specific-antibody of the pcDNA-PCV2-ORF2 DNA vaccine group was significantly higher than that of the control group (P＜0.05).The number of CD~3+ T-lymphocytes of CpG adjuvants group was significantly higher than that of the control 7 days after vaccination. The number of CD~4+ and CD~8+ T-lymphocytes of experiment groups was higher than that of the control group 35 days after vaccination. Humoral immune response was significantly higher than control group. But to cytokine group, no significant difference between these experiment group. Piglets immunized with higher dose (500μg) of DNA vaccine demonstrated higher humoral and cellular immune responses than piglets immunized with lower dose (250μg) of DNA vaccine. The results demonstrated that two DNA vaccines could induce good humoral and cellular immune responses and it showed some dose-dependent character.As the adjuvant, 5 kinds of pig interleukin immuned the piglets by the routes of co-injection with pcDNA-PCV2-ORF2 DNA vaccine, PBS and pcDNA3.1 (+) as controls. Fluorescence Activated Cell Sorter (FACS) was used to detect the number of CD~3+, CD~4+ and CD~8+ T lymphocytes and indirect ELISA methods were employed respectively to detect the celluar immune and anti-PCV2 antibody. The experiment group vaccinated the DNA vaccine with pig IL-2 ,IFN-γshowed higher cellular immune responses than that vaccinated with DNA vaccine alone and the number of CD~3+ T lymphocytes was peaked at 35 day. The group vaccinated the DNA vaccine with IL-4, IL-6, and IL-6/4 showed higher humoral immune responses than that vaccinated with DNA vaccine alone. IL-6 as adjuvant enhanced immunocity of the humoral responses to DNA vaccine and produced highly level of specific-antibody. The results demonstrated that CpG, IL-2, IL-4, IL-6, IL-6/4 and IFN-γwere good adjuvants which could significantly enhance the immunogenicity of pcDNA-PCV2-ORF2 DNA vaccine. The adjuvants immunocity of CpG and IL-6 was better than IL-6/4, IL-2 and IFN-γ.The result showed that CpG and interleukin as adjuvants have good prospective to DNA vaccine. And the study on adjuvant immunocity to PCV2 DNA vaccine was first report in the world. |
