The Construction And Immunogenicity Study Of Recombinant CAV-2 And DNA Vaccine Of GP5 And M Of Porcine Reproductive And Respiratary Syndroma

GP5 and M protein encoding genes from porcine reproductive and respiratory syndrome virus Jilin strain were amplified using the special primers, and cloned into pMD18-T vector, generating pMD18T-G,pMD18T-M,respectively, which were further sequenced and phylogenetic analysis. The results indicated that GP5 gene contains 603-bp encoding 200 amino acids, and M gene contains 525-bp encoding 175 amino acids. The homology of GP5 with PRRSV American strain was 77.7-99.7% in nucleotide, and 81.3-98.9% in amino acids. The homology of M with PRRSV American strain was 91.4-99.8% in nucleotide and 92.1-99.7% in amino acids. The phylogenetic analysis demonstrated that PRRSV Jilin strain belonged to standard virulent strain such as 01NP strain, BJ-4 strain and RV-2332 strain, as well as American strain, but was different from Erope LV strain, indicating that PRRSV Jilin strain belongs to American strain. PRRSV Jilin strain have the same antigen epitopes and antigen as BJ-4 strain and RV-2332 representative strain, and PRRSV Jilin strain, which provide the foundation for development of nucleic acid vaccine and recombinant adenovirus vaccine.Construction of eukaryotic expression vectors of GP5 and M. GP5 and M genes were recovered from pMD18T-GP5 and pMD18T-M, and subcloned into pIRES-neo vector digested by EcoRV/BamHI and XbaI/BamHI, respectively, resulting to pIRES-GP5 and pIRES-M, which were used as gene vaccine for PRRSV. The mice were divided into five groups and immunized with pIRES-neo, pIRES-GP5, pIRES-M, pIRES-G+pIRES-M and inactivated vaccine, respectively. The antibodies response level was evaluated by ELISA and serum neutralization test, and cellular immune response was assayed by lymphocyte transformation test. The results indicated that all immunity groups could produce special humoral and cellular immunity in which the mixed immunity group of pIRES-GP5 and pIRES-M could developed better immune response than the other groups, but less than the inactivated PRRSV group.Construction of recombinant canine adenovirus type-2 expressing GP5 and M protein from PRRSV. GP5 and M genes were recovered from pMD18T-GP5 and pMD18T-M, and subcloned into pEGFP-C1, producing pEGFP-GP5 and pEGFP-M, from which the expression cassettes of GP5 and M were excised with AseⅠ/MLuⅠand cloned into SspⅠsite of pVAX-E3, generating pVAX-GP5 and pVAX-M. Plasmids pPoly-Ⅱ-CAV-2-GP5 and pPoly-Ⅱ-CAV-2-M were constructed by cloning the NruⅠ/ SalⅠfragments of pVAX-GP5 and pVAX-M into NruⅠ/ Sal site of pPolyⅡ-CAV-2. The genome containing canine adenovirus was released from plasmids pPoly-Ⅱ-CAV-2-GP5 and pPoly-Ⅱ-CAV-2-M with PmeI and AscI, respectively, and were transfected into MDCK using Lipofectamine 2000TM. 4μg purified recombinant genome dissolved in 300μl DMEM and 25μl Lipofectamine 2000TM dispersed in 300μl DMEM were mixed up at room temperature for 20 min, supplemented with 1.4 ml DMEM and then overlaid onto the 80% confluent MDCK cells. After 12 h of incubation, the medium was replaced by a complete DMEM containing 5% newborn calf serum. Transfected cells were left at 37℃and propagated for days until cytopathic effect (CPE) appeared. The recombinant virus genomes were extracted and further identified by restriction enzyme , PCR, RT-PCR and Western blotting, demonstrating that the two recombinant viruses can transcript mRNA of GP5 and M protein, and can express the proteins, and have good genetic stability.The piglets were immunized intramuscularly with CAV-2-GP5 and CAV-2-M and the immune responses were evaluated by Western blot, neutralizing antibody assay, ELISA and peripheral blood lymphocyte proliferation response, indicating that the two recombinant viruses could induce special antibodies against GP5 and M proteins, and develop special humoral and cellular immune responses, and the mixed viruses induce higher immune responses than the single recombinant virus.