| Bovine viral diarrhea(BVD)and bovine infectious rhinotracheitis(IBR)are two major diseases that endanger cattle and other ruminants,the pathogens of the two diseases are bovine viral diarrhea virus(BVDV)and bovine infectious rhinotracheitis virus(IBRV).BVDV and IBRV often infect cattle herds,causing some clinical symptoms such as diarrhea,hemorrhagic syndrome,abortion,pneumonia,encephalitis,rhinitis,and ophthalmia,which have brought huge economic losses to the cattle industry.At present,the prevention and control of these two kinds of diseases mainly rely on inactivated vaccines or attenuated vaccines.Unfortunately,there are many shortcomings such as short duration of immunization,easy reversion of virus virulence,and high storage and transportation costs.Therefore,nucleic acid vaccines with the advantages of long-lasting immune response,simple preparation process,and safe application have become the hotspots of vaccine development.In this study,the dominant antigen genes of BVDV and IBRV were inserted into the pVAX1 eukaryotic expression vector to obtain a recombinant vector capable of expressing both antigen genes and evaluate its immune effect.The aim was to develop a nucleic acid vaccine that can effectively prevent BVD and IBR.Main tasks as follows:1.Cloning and bioinformatics analysis of BVDV EO and IBRV gD target genes:Primers were designed according to the BVDV and IBRV reference sequences included in GenBank,and the BVDV E0 gene and IBRV gD gene were amplified by PCR and sequenced;the target protein was analyzed using bioinformatics methods.The results showed that the amplified fragments size of BVDV E0 gene and IBRV gD gene were same as expected,and their proteins had good immunogenicity,which could be used as candidate antigen genes for nucleic acid vaccines.2.Construction of eukaryotic expression vector PVAX1-E0-IRES-gD:IRES sequence was amplified by PCR;the IRES,BVDV E0,and IBRV gD gene fragments were sequentially ligated into the pVAX1 vector to construct the pVAX1-E0-IRES-gD recombinant plasmid The recombinant plasmid was identified by double digestion and DNA sequencing.The results showed that the eukaryotic expression vector pVAX1-E0-IRES-gD required for the study was successfully constructed.3.Expression assay of PVAX1-E0-IRES-gD in vitro:The plasmid pVAX1-E0-IRES-gD was transfected into 293T cells by liposome method.After 48 hours,primary antibodies(BVDV,IBRV Positive serum)and secondary antibodies(Rabbit Anti-bovine IgG-FITC)were used for indirect immunofluorescence detection.The results showed that green fluorescence could be observed under the fluorescence microscope,which proved that the target proteins E0 and gD were successfully expressed in 293T cells and had good reactionogenicity.4.Study on immune effect of bivalent vaccine pVAX1-E0-IRES-gD:Mice were immunized with pVAX1-E0-IRES-gD DNA vaccine at different doses and boosted once after 14 days of first immunization.The ELISA method was used to detect the anti-E0,gD antibody levels and the IFN-y,IL-4 content in the mouse serum,and the MTT method was used to detect the mouse spleen lymphocyte proliferation ability.The results showed that in terms of antibody and cytokine levels and the proliferation of spleen lymphocytes,the nucleic acid vaccine group was significantly higher than the empty vector and the negative control group,and the differences were extremely significant,the high-dose immunization group is better than the medium and low dose groups;in addition,the high-dose nucleic acid vaccine immunization group was not significantly different from the BVD-IBR inactivated vaccine group.This study successfully constructed the BVD-IBR bivalent DNA vaccine.The in vitro expression test proved that the target protein has good reactionogenicity,and animal immunity tests proved that it can stimulate the body to produce a good immune response.The above test results provided reliable data and theoretical support for the development of BVD-IBR bivalent DNA vaccine. |
PDF Full Text Request