| 【Objective】Cytokine immune adjuvant effect , by slow and sustained release in the body , thereby enhancing the immune function of the vaccine. Animal interleukin -7 (IL-7), a multifunctional cytokine, interleukin -7 as an important member of the interleukin family, it is not only plays an important role in B- and T-cell generation and development and in improvement of animal immune capacity coordinating with other cytokines. In order to verify the interleukin 7 gene DNA vaccine enhanced role, The study useing canine parvovirus of VP2 DNA vaccine and recombinant canine interleukin -7(cIL-7) DNA vaccine common immune mice, to investigate the immune enhancing effects of canine IL-7 (cIL-7) gene co-immunized with expression vectors of canine parvovirus VP2 DNA vaccines in mice.【Method】First the canine spleen lymphocytes were isolated from the canine spleen, and with con A stimulated, canine total RNA was extracted by Trizol, and flipped into cDNA, Canine IL-7 genes, both with stop codon and without stop codon, were amplified from canine splenic lymphocytes by RT-PCR, and then were inserted into the eukaryotic expression vector pcDNA3.1A to contruct the non-fused and Myc/His-tag-fused cIL-7 eukaryotic secretory expression vectors (pcDNA-cIL7 and pcDNA-cIL7/MH), respectively. To determine whether the expression vector could mediate cIL-7 gene expression in eukaryotic cells, the pcDNA-cIL7/MH plasmid was transfected into HEK293T cells using calcium phosphate transfection method for transient expression. The mice were co-immunized with VP2 gene expression vector (pcDNA-CD5-VP2, constructed previously in our laboratory) and cIL-7 gene expression vector pcDNA-cIL7, the mice were immunized with pcDNA 3.1, pcDNA-cIL7 and pcDNA-CD5-VP2 as a control. Immunized mice with the intramuscular method, DNA plasmids DNA extraction of endotoxin-free kit, and plasmid DNA injected into mouse hind leg muscles. Mice were immuned twice, the second immunization in the 14 days after the first immunization. After immunization, before two immune 2 days and after first immune 35 days, taked blood from the corner of mice eye, and separated serum. the antibodies against CPV in the immunized mice at different time were measured by ELISA. The spleen lymphocyte proliferation response at 35 d post-immunization was determined by lymphocyte proliferation assay, and the interferon-γexpression level of the mouse lymphocytes was measured by ELISA.【Results】The results showed that the sequence of cIL-7 gene amplified was consistent with that in GenBank. Western-blot showed that recombinant cIL-7 could be expressed and secreted in HEK293T cells. Immunization results showed that the antibody levels and the neutralizing antibody titers in the serum of VP2/cIL-7-immunized mice were significantly higher than that of VP2-immunized mice (P <0.01 and P <0.05), respectively. The lymphocyte stimulation indexes and secreted IFN-γlevels of the VP2/cIL-7-immunized mice were significantly higher than that of VP2-imunized mice (P <0.05), respectively.【Conclusion】The cIL-7 gene can significantly enhance the immune response of the mice to CPV VP2 DNA vaccine. |
PDF Full Text Request