Study On Bivalent Genetic Engineering Vaccine And DNA Vaccines Against Foot-and-mouth Disease And Pseudorabies

Foot-and-mouth disease virus(FMDV) is the causative agent of Foot-and-mouth disease(FMD),which is a highly infectious and one of the most important animal viral diseases affecting cloven hoofed animals in the world.It can jeopardize the animal husbandry and affect countries to participate in the international trade of animals and animal products.It ranks the first one in the List A of animal infectious diseases according to the Office International des Epizooties(OIE) and Food and Agriculture Organization (FAO).FMDV is one of the most important pathogens involved in the Global Animal Disease Eradication Program and the Biological Weapons Convention(BWC).Also,it belongs to the ten most important animal pathogenic microorganisms in China.FMDV is a member of the Aphthovirus genus in the Picornaviridae family.Seven serotypes(A,O, C,Asia 1,and South African Territories 1,2,and 3) have been identified,and multiple subtypes can be identified under several serotypes.There is no cross protection between different serotypes.Serotype O is the most widespread virus in the world including Africa, South Asia and Middle East.In China serotype O is the prevalent serotype as well. Vaccination is the significant measure to control and eliminate FMD.Although some achievements have received in prevention of FMD by using inactivated vaccines of serotype O,the vaccines must be improved.In the present study,two recombinant pseudorabies virus(PRV) strains TK~-/gE~-/VP22-P1 and TK~-/gE~-/VP22-P1/2A/3C co-expressing the VP22 gene of bovine herpes virus 1(BHV-1) and P1 gene or P1/2A/3C gene of FMDV,respectively,were constructed.At the same time,two DNA vaccines pcDNA-VP22-P1 and pcDNA-VP22-P1/2A/3C were constructed as well.The immune effect of these vaccines were evaluated and compared in a Balb/c mouse model.1.Cloning and sequence analysis of VP22 gene of BHV-1,and P1 and P1/2A/3C genes of FMDVIn order to fuse the VP22 gene in-frame to the 5'-end of P1 gene and P1/2A/3C gene respectively,the VP22 coding sequence without the stop codon was amplified from a recombinant plasmid carrying the VP22 gene using a sense primer with a HindⅢrestriction site and an antisense primer with an EcoRV restriction site.The P1 and P1/2A/3C coding sequence without the start codon were separately amplified from the corresponding recombinant plasmids using sense primers with a EcoRV restriction site and antisense primers with XbaⅠrestriction site.All the PCR products were cloned into the cloning vector pMD18T,resulting in recombinant plasmids pMD18-T-VP22, pMD18-T-P1 and pMD18-T-P1/2A/3C respectively.DNA sequence analysis demonstrated that the sequences of the three genes had 100%identity with the sequences published.2.Construction and evaluation of DNA vaccines co-expressing VP22 and P1 or P1/2A/3CTo construct DNA vaccines pcDNA-VP22-P1 and pcDNA-VP22-P1/2A/3C co-expressing BVP22 with P1 or P1/2A/3C respectively,the VP22 fragment was released from pMD 18-T-VP22 by HindⅢand EcoRV digest,and then cloned between the HindⅢand EcoRV sites of pcDNA3.1(+),resulting in recombinant plasmid pcDNA-VP22.The P1 and P1/2A/3C fragments were released from pMD18-T-P1 and pMD18-T-P1/2A/3C respectively by EcoRV and XbaⅠdigest,and then subcloned between the EcoRV and XbaⅠsites of pcDNA-VP22,resulting in recombinant plasmids pcDNA-VP22-P1 and pcDNA-VP22-P1/2A/3C respectively.To evaluate the immune effect of the DNA vaccines pcDNA-VP22-P1 and pcDNA-VP22-P1/2A/3C,the two DNA vaccines were compared with PBS,pcDNA3.1, pcDNA-VP22,pcDNA-P1,FMDV inactivated vaccine,pcDNA-VP22-P1 combined with FMDV inactivated vaccine,and pcDNA-VP22-P1/2A/3C combined with FMDV inactivated vaccine in the Balb/c mouse model.The results showed that neutralization and ELISA antibodies against FMDV could be detected at 14 and 28 days post the first immunizaction with the four DNA vaccines including pcDNA-P1,pcDNA-P1/2A/3C, pcDNA-VP22-P1 and pcDNA-VP22-P1/2A/3C.The levels of FMDV antibodies induced by the DNA vaccines carrying the VP22 gene were significantly higher than those induced by the DNA vaccines without the VP22 gene(P＜0.05).The mice co-vaccinated with the DNA vaccine and FMDV killed vaccine generated higher levels of FMDV antibodies than those vaccinated with the DNA vaccine alone(P＜0.05),but similar to those vaccinated with FMDV killed vaccine(P＞0.05).The results of lymphocyte proliferation assays revealed that DNA vaccine could induce a better cell immunity than FMDV inactivated vaccine(P＜0.05).3.Construction and evaluation of the bivalent recombinant vaccine strains TK~-/gE~-/VP22-P1 and TK~-/gE~-/VP22-P1/2A/3CRecombinant transfer vectors pIECMV-VP22-P1 and pIECMV-VP22-P1/2A/3C were constructed using the PRV universal transfer vector pIECMV.PRV TK~-/gE~-/LacZ~+ was used as the parent strain,the recombinant PRV TK~-/gE~-/VP22-P1 expressing VP22-P1 fusion gene and recombinant PRV TK~-/gE~-/VP22-P1/2A/3C expressing VP22-P1/2A/3C fusion gene were obtained by plaque purification,PCR and southern blot identification.Then the recombinant viruses were purified.The co-expression of the foreign gene VP22-P1 and VP22-P1/2A/3C in recombinant PRV strains were confirmed by Western blot analysis.The results showed that recombinant PRV TK-/gE-/VP22-P1 and PRV TK~-/gE~-/VP22-P1/2A/3C were successfully constructed.At the same time the hereditary stability and proliferation titres of the recombinant PRV strains TK~-/gE~-/VP22-P1 and TK~-/gE~-/VP22-P1/2A/3C were detected.The immune efficacy of both recombinant PRV strains was evaluated and compared with the FMDV inactivated vaccine in a Balb/c mouse model.The results showed that the levels of FMDV antibodies induced by either strain TK~-/gE~-/VP22-P1 or strain TK~-/gE~-/VP22-P1/2A/3C were similar to those induced by the FMDV inactivated vaccine(P＞0.05).This indicated that both fusion proteins VP22-P1 and VP22-P1/2A/3C could stimulate strong immune response to FMDV.In the present study,both DNA vaccines and recombinant PRV vaccines which co-express the VP22 gene of bovine herpes virus 1 and the P1 gene or P1/2A/3C gene of FMDV were successfully constructed.Animal experiments were carried out to evaluate the immune effect of these vaccines,and to compare with the traditional inactivated vaccine of FMDV.Our data will contribute to the development of a novel generation of vaccine against FMDV.