Cultivation Of Vero Cells Adapted Goatpox Virus And Preparation Of Monoclonal Antibody Against P32 Protein Of Goatpox Virus

GTPV(Goatpox virus,GTPV)is a contagious and acute infectious disease which can infect of all ages,sex and species of goats causing fever,skin pimples and severe pathological changes.It led to the decrease of the production capacity Since GTPV is a seriously threaten to the development of sheep industry in our country.Recently,attenuated vaccine and inactivated vaccine are commonly used to prevent and control this disease.So,it is important to establish a rapid,efficient,convenient and cheap detecting method.In this study,the GTPV-XZ strains isolated from LT cells were cultured in Vero cells.At the same time p32 protein of GTPV was expressed and preparation of monoclonal antibody prepared which lays a foundation for establishing a rapid,efficient,convenient and cheap detecting method.1.Cultivation of Vero cells adapted Goatpox VirusThe GTPV was transferred to Vero cells for consecutively passage.After passaging 20 generations,CPE caused by GTPV appeared stable.After incubating for 3 days,cells appeared shrinking and formed clumps.After incubating for 5 days,drop of cells was observed.Viruses were harvested when CPE was around 80%and kept on passaging.Virus titers of each generation of virus were determined using both methods of TCID50 and qRT-PCR.The result showed that virus titers on Vero cells in earlier stage appeared relative low.After passage for 20 generations,virus titers became stable and slight higher than that of previous generations.So the Vero cells adapted goatpox virus was acquired.2.Prokaryotic expression of P32 gene of GTPVP32 gene of GTPV was amplified and inserted into prokaryotic expression vector pET-32a.After transferred to BL21,the protein was expressed.SDS-PAGE showed that a great quantity of P32 proteins could be expressed in soluble form under the condition of 37?,220 rpm and 1M IPTG.After purification,the concentration of recombinant protein is 2.5 mg/mL.BALB/c mice were immunized with purified recombinant P32 protein for antiserum preparation,the result of IFA showed that the purified recombinant P32 protein had a good immunogenicity,which provides material for the preparation of monoclonal antibodies against P32 protein.3.Preparation of monoclonal antibodies against P32 protein of GTPVBALB/c mice were immunized with purified recombinant His-P32 fusion protein.Spleen cells of immunized mice were fused with myeloma cell(SP2/0)after forth immunization.ELISA and IFA were used to detect the positive cell.Finally,two monoclonal antibodies(McAbs)were screened and named 2B4,2E10.The result of IFA showed that specific fluorescence was observed when Vero cells infected with GTPV.Also,western blot confirmed that both 2B4 and 2E10 McAb could react with GTPV and showed specific band.Besides,the two McAbs have no reaction to 2 ORFVs isolated previously by our lab.Therefore,a fast and sensitive antigen detection method based on the two McAbs can be established.