Preparating The Monoclonal Antibodies Of Porcine Circovirus Type 2 And Constructing The Multicopies P28-ORF2 Recombinant Of Swinepox Virus

Porcine circovirus infection is a swine infectious disease caused by porcine circovirus type 2(PCV2),which will cause huge weight loss,physical decline and difficulty of breathing.Capsid protein(Cap protein)is a major immunogenic protein in PCV2.At present PCV2 subunit vaccine on the market is mainly based on eukaryotic expression vector: such as baculovirus vector expression system.Swinepox is a swine infectious disease caused by Swinepox virus(SWPV),which is characterized by pox and scabby formed on the skin.The majority of infected pigs can be self-rehabilitation.SWPV is an ideal genetic engineering vaccine vector,for the reasons that it has a large genome(146kb),contains multiple replication nonessential areas-with strict host specificity,which can carry multiple fragment of exogenous gene.In this study,we used swinepox virus as the vector,which based on the ORF2 gene of PCV2 JX-CZH strain,constructed eight recombinant plasmids containing different copy numbers of P28-ORF2.Eight recombinant swinepox viruses were obtained by homologous recombination technique.The PCV2 monoclonal antibody was screened to explore the relationship between the different copy numbers of P28-ORF2 and the Cap protein content of recombinant swinepox virus1.The preparation of PCV2 monoclonal antibody.In this study,we used swinepox virus as the vector,which based on the ORF2 gene of PCV2 JX-CZH strain,obtained the recombinant swinepox virus expressing PCV2-Cap by homologous recombination technique.We used the mice of antigen immunized BALB/c,which immunized by the recombinant swinepox virus obtained four PCV2 monoclonal antibodies screened by hybridoma technique and His-Cap protein expressed in PK15 cells.Western-blotting and immunofluorescence assay showed that four monoclonal antibodies could not only be used in Western-blotting of PCV2-Cap,but also could specifically bind to PCV2 virus particles and used in immunofluorescence and immunohistochemical staining of PCV2.The four PCV2 monoclonal antibodies obtained in this study laid the foundation for the establishment of qualitative and quantitative methods for PCV2-Cap in the future.2.Constructing the Multicopy P28-ORF2 recombinant of swinepox virusIn order to understand the relationship between the different copies of P28-ORF2 and the expression content of PCV2-Cap in the swinepox virus genome.We used the swinepox virus as the vector,constructed 8 different copy number(1-8 copies)of P28-ORF2 recombinant swinepox viruses,and determined the microbiological characteristics of 8 recombinant swinepox viruses.(1)The size of the fluorescent spot(330 ?m)of the recombinant virus and TCID50(about 10-12/0.1mL)showsthat:The deletion of P28-ORF2 in the genome of swinepox virus has no effect on the proliferation of recombinant swinepox viruses in PK15 cells.(2)Electron microscopic observation shows that the recombinant swinepox viruses of different copy numbers are consistent with the parental virus in phological and structure.(3)Western-blotting results: 1 copy of PCV2-Cap expression is the lowest.With the increase of P28-ORF2 copy number,PCV2-Cap expression also increases,4 copies of P28-ORF2 reaches the highest expression of PCV2-Cap.The results of this study shows that the multi-copy recombinant swinepox viruses can significantly increase the expression of exogenous protein based on the expression of exogenous protein in the swinepox virus vector,and the 4 copies are the appropriate copy number.