Canine GM-CSF Gene On The Canine Parvovirus Of VP2 DNA Vaccine Enhancement

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is secreted by T cells, B cells, endothelial cells, fibroblasts, macrophages. GM-CSF is kind of important hematopoietic growth factor and immune regulating factor which could improve the survival, proliferation, differentiation of precursor cell and differentiation, colonies formation of granulocytes and monocytes/macrophages. GM-CSF could attracted dendritic cells, monocytes and other antigen-presenting cells to the antigen injection position and plays a key role in the differentiation of precursor cells. Therefore, GM-CSF is considered a new high molecular adjuvant. VP2 protein, encoded by canine parvovirus (CPV) genome, is a major structural protein and epitope. The DNA vaccine derived from the VP2 gene can stimulate the immune responses to CPV. To enhance the VP2 gene vaccine potency, the dog granulocyte and macrophage colony-stimulating factor (GM-CSF) gene was used as a biological adjuvant.GM-CSF gene was amplified by RT-PCR from dog spleen lymphocytes and connected with pcDNA3.1A vector to construct two GM-CSF eukaryotic expression vectors respectively: non-fused vector pcDNA-cGM-CSF and Myc/His-fused vector pcDNA-cGM-CSF/MH. Before immunization, the pcDNA-cGM-CSF/MH vector was transfected into HEK293T cells for transient expression to determine whether the recombinant cGM-CSF could be expressed in the eukaryotic cells in a secretory manner. The mice were separately immunized with VP2 vector (pcDNA-CD5-VP2, constructed previously in our laboratory) and VP2/cGM-CSF combined vectors (pcDNA3.1 vector as a negative control). After immunization, the antibodies against CPV from the immunized mice serum were measured by ELISA。The spleen lymphocyte proliferation response was measured by lymphocyte proliferation assay, and the interferon-γlevel of the mouse lymphocytes was measured by ELISA kit.The results showed that the recombinant cGM-CSF could be secretively expressed in HEK293T cells. The antibody titer of the mice co-immunized with VP2 and cGM-CSF vectors was significantly higher than immunized with VP2 vector only (P <0.01). The lymphocyte stimulation index of VP2/cGM-CSF co-immunized mice was significantly higher than VP2 only immunized mice (P <0.05). The IFN-γlevel of lymphocytes from the co-immunized mice was significantly higher than the VP2 immunized mice (P <0.01). In conclusion, cGM-CSF gene can significantly improve the CPV VP2 DNA vaccine immunogenecity.