Construction And Immune Effects Of Recombinant Pseudorabies Virus Expressing VP2 Gene Of Porcine Parvovirus

Porcine parvovirus(PPV),a member of the genus Parvovirus and the family Parvoviridae,was demonstrated to be a significant causative agent in porcine reproductive failure characterized by mummification,abortion,stillbirth and neonatal death.PPV was initially found as a polluting small DNA virus while cultivating swine fever virus,then gradually came to realize that it is an important cause of death in weaned pigs and embryonic loss.The organization of PPV consists of single-stranded negative-sense DNA of 5000 nucleotides.The PPV genome includes two open reading frames(ORFs).ORF1 encodes the nonstructural proteins NS1,NS2,and NS3;ORF2 encodes the structural proteins VP1 and VP2.The VP2 protein of PPV encompasses major antigenic domains with the capacity to induce immune response.PPV is an economically important viral disease causing serious economic losses in the swine industry worldwide.In 1980 s,several PPV strains have been isolated in China,which have proven to be a crucial viral agent associated with reproductive failure.Pseudorabies virus(PRV)is another significant causative agent in porcine reproductive failure,resulting in nervous disorders,stillbirth,abortion and boar infertility as symptoms.The genome of PRV is a linear DNA molecule of about 150 kb,which includes many nonessential genes which foreign genes can be stably inserted,making the attenuated PRV a promising candidate for the development of a live vaccine vector to confer protection against both PRV and other diseases.In this study,we have expressed the major immunogenic protein VP2 of PPV using the attenuated PRV as parental strain,and the recombinant virus p GVP2 was successfully generated,which provides effective support forPRV and PPV or co-infection both in theory and technology.In order to develop the recombinant attenuated PRV expressing PPV VP2,the VP2 gene of PPV was amplified by PCR using specific primers with the restriction enzyme cutting site Bam HI,and then the sequence was purified and analysised.We digest vector p G and plasmid VP2 with Bam HI and then recoveried them.After dephosphorylation of the digested vector p G,we linked the VP2 gene into the vectorPG and got the recombinant plasmid named p GVP2,and we confirmed the success of the construction of the transfer plasmid with biological technology of PCR,restriction enzyme digestion and sequencing.The genome of PRV attenuated vaccine and the transfer plasmid were transfered into ST cells.The recombinant virus was subjected to five cycles of plaque purification and identified by PCR.The virus replication assay of the recombinant rPRV PGVP2 on multiple cells indicated that no obvious difference was detected between the recombinant and the parent viruses.The study also showed that ST cell present better adaptation in consideration of its higher virus titers than VERO orPK-15 cells.Furthermore,the virus replication of the recombinant rPRV PGVP2 on ST cell was examined by growth curve analysis.The virus titer was continuously increases in both lysate and pellet of infected cells with more abundant in the pellet at 10 h.p.i.and towards equality thereafter at 10 h.p.i to12 h.p.i..Then the numerous recombinant virus particles released into the supernatant at 12 h.p.i,and gradually stabilized in both lysate and pellet at 36 h.p.i..The research on safety testing indicated that the immunization of the recommbinant irus was secure for mice.Pass-generation test demonstated that the heredity character of the recommbinant rPRV PGVP2 was stable.The expression of the inserted gene in the recommbinant virus was confirmed by RT-PCR and Western blot.The study for the recombinant virus on partial biological characteristics lends further support to the development of bivalent vaccines and in particular,against PRV and PPV.In order to evaluate the immunogenicity of the recombinant rPRV PGVP2 virus,we immunized intramuscularly into the quadriceps muscle of 6-week-old female Kunming mice with attenuated PRV parent strain,commercial inactivated vaccine of PPV,recombinant virus,DMEM culture solution,and boosted them with an equivalent dose 2 weeks later.Serum samples were collected after the first immunization to identify the humoral immune condition in mice each week by ELISA.Five mice of each group were killed 8 weeks later after the first immunization to evaluate the level of lymphocyte cell subsets.To investigate the degree of protection elicited by rPRV PGVP2 and the parent PRV,the remaining five mice from each group were challenged with the virulent PRV strain.The result in rPRV PGVP2 group showed that the level of VP2-specific humoral immune response was low for the first immunization but significant for the second immunization.The recombinant virus elicited significant cellular immune response towards the determination of T lymphocyte subsets.The challenge assay showed that groups of recombinant virus and attenuated PRV parent strain could successfully protect the mice against the virulent PRV challenge.These results demonstate that the recombinant virus can be an excellent potential vaccine.