Isolation And Identification Of Bovine Viral Diarrhea Virus And Research On Combined Immune Of E0 DNA Vaccine And Cytokine Adjuvant

Bovine viral diarrhea virus(BVDV/MD) is a member of the pestivirus genus in the virus family Flaviviridae, mainly causes bovine viral diarrhea(BVD) with worldwide distribution. Because of reproductive obstacle and persistent infection, BVD causes a significant losses in cattle farming worldwide. At present, there is no commercial vaccines to prevent the disease, Therefore, the urgent need to develop new vaccines over the prevention and control of the disease. E0 BVDV structural proteins highly conserved, on which a neutralizing epitopes. Therefore, this study aimed at local strain E0 gene of BVDV-TC and eukaryotic gene expression plasmid constructed two cytokines, to study the DNA vaccine and cytokine adjuvant combined immune effects.In this study, isolated bovine viral diarrhea-XJ named BVDV-TC were respectively amplified by RT-PCR the E0 gene and bovine of Bovine granulocyte- macrophage colony stimulating gene fragment inserted into the vector pVAX1 eukaryotic expression vector. Then liposome 2000 each of the recombinant plasmids were transfected to BHK-21 cells for transient expression, RT-PCR and Western blot analysis experiments showed that the eukaryotic recombinant E0 gene expression plasmids were successfully transfected into BHK-21 cells and expressed; MTT experiments show that the culture of cytokines recombinant plasmid transfected cells can stimulate lymphocyte proliferation, with good biological activity.To healthy mice were immunized as follows recombinant plasmids:Only injection pVAX1-E0 and pVAX1-E0 respectively Laboratory to build good pVAX1-IL-2; pVAX1-GM-CSF; pVAX1-GM-CSF and pVAX1-IL-2 co-immunized mice, and the empty vector pVAX1 as a control group. Improved by indirect ELISA and MTT assay immunized mouse serum antibodies specific E0 and spleen lymphocyte proliferation levels compared with the control group, E0 vaccination antibody levels eukaryotic recombinant plasmid and mouse lymphocyte proliferation levels have(P<0.01).The pVAX1-IL-2 and pVAX1-GM-CSF co-immunization group showed a stronger immune stimulating activity.This study successfully constructed bovine viral diarrhea disease DNA vaccine and cytokines adjuvants can stimulate the production of cellular and humoral immunity, and has a significant immune adjuvant effect. New and efficient for the development of this DNA vaccine prevent bovine viral diarrhea disease provides a theoretical and experimental data.