| Porcine circovirus type 2 (PCV2) is the primary pathogen of postweaning multisystemic wasting syndrome (PMWS) and other associated conditions in pigs. In this study, several mammalian recombinant plasmids, expressing porcine granulocyte-macrophage colony-stimulating factor (GM-CSF), PCV2 capsid protein (CP), and co-expressing CP and GM-CSF, were constructed respectively. The humoral and cellular immune responses elicited by recombinant plasmids expressing PCV2 CP were analyzed in mice and pigs; moreover regulating functions of recombinant plasmids expressing porcine GM-CSF and IL-2 to PCV2 DNA vaccine , respectively, was evaluated at the same time.The cDNA of porcine GM-CSF gene was amplified from the peripheral blood lymphocytes (PBL) of swine by RT-PCR and was used to construct a recombinant eukaryotic expression plasmid pcDNA3.1-gm-csf. ORF2 gene of PCV2 was obtained by PCR and three recombinant plasmids including pcDNA3.1-orf2, pcDNA4-orf2 and pIRES-orf2/gm-csf co-expressing CP & GM-CSF were constructed respectively. It was testified that CP gene in pcDNA3.1-orf2, pcDNA4-orf2 or pIRES-orf2/gm-csf, GM-CSF gene in pcDNA3.1-gm-csf and pIRES-orf2/gm-csf could be expressed in COS7 cells by indirect immunofluorescene assay and colony-stimulation forming assay.The humoral and cellular immune responses induced by the recombinant plasmids pcDNA3.1-orf2 and pcDNA4-orf2 were analyzed in mice. The results showed that after three intramuscular injections, specific antibodies to PCV2 were present in serum of 80% mice with pcDNA3.1-orf2 and 100% mice with pcDNA4-orf2; the splenocytes proliferation of mice immunized with recombinant plasmids, stimulated by concanavalin A (ConA) or recombinant PCV2 CP, were distinctly increased, and the lysis activities of CTL and NK cells were significantly elevated; pcDNA4-orf2 arose relatively lower cellular immune responses in comparison with pcDNA3.1-orf2 recombinant plasmids;.the number of CD3+CD4+CD8- and CD3+CD4-CD8+ T lymphocytes sub-populations of splenocytes rose after the mice were immunized with recombinant plasmids; On the other hand, there were no differences in immune responses induced by intramuscular and intranasal administration of pcDNA3.1-orf2.7-day-old healthy piglets were randomly divided into 5 groups which were blank vector (group I), pcDNA3.1-orf2 (group II), pcDNA3.1-orf2 plus pcDNA3.1-gm-csf (group III), pIRES-orf2/gm-csf (group IV), and pcDNA3.1-orf2 plus pcDNA3.1-IL-2 (encoding porcine interlukin-2) (group V). All piglets were injected intramuscularly three times at 2-week intervals. The results showed that all groups of pigs immunized with recombinant plasmids could produce low-level specific ELISA antibody to PCV2 and neutralizing antibodies with a average titre 1:6.7 (group II), 1:29.3 (group III), 1:26.7 (group IV) and 1:20.2 (group V) respectively; the proliferation activities of PBL from pigs in Group IV and V were higher than those of pigs in Group II and III; meanwhile, lysis activities of NK cells of pigs in Group III, IV and V clearly exceeded Group II; the number of Th, Tc, and m/aTh of pigs in each group vaccinated with recombinant plasmids, especially in Group V, were elevated compared with those inGroup I; level of IL-4 expressed in PBL of pigs in all groups immunized with recombinant plasmids was increased in comparison with those in Group I. Meanwhile, the expression level of IL-12 in group IV and V was higher than those in Group II and III. At weeks 4 after the third immunization, all pigs were challenged with PCV2 BJ-HB strain via intranasal and intraoral routes. The results demonstrated that compared with Group I, all pigs from each group immunized with recombinant plasmids did not develop pyrexia, the viral excretion in nasal and rectum swabs and the percentage of viraemia were significantly decreased, especially in Group V; the ratio of PCV2 positive tissues from Group V detected by PCR and immunohistochemistry assay was the lowest. These results indicated that protective efficacy of combined vaccination of pcDNA3.1-orf2 and pcDNA3.1-IL-2 was super... |
PDF Full Text Request