Selection Of Cell Adaptable Virus Of Very Virulence Goose Paravirus And Prokaryotic Expression Of VP3 Gene

Six strains viruses were isolated by goose embryo inoculation with tissue samples from death geese, which supposed to be infected by goose paravirus (GPV). These viruses could not agglutinate red cells and not cause embryo die. The viruses were identified as GPV by antibody-sandwich ELISA based on monoclonal antibody. One (GPV-CZ) of six strains GPV was characterized by PCR, electronic microscope and ELD50 assay. The results indicated that ELD50 of the virus was 10-3.1/0.2ml, non-enveloped round virions with diameter about 20nm under electronic microscope.GPV-CZ strain was passaged in goose embryo fibroblast(GEF)and detect by indirect immunofluorescence assay. The results showed that the virus adapted the cells after the ninth generations(named as GPV-CZ-ca) and immunofluorescence could be found at the tenth passage in GEF with monoclonal antibodies to GPV. The earliest time point of the cell adaptable virus in GEF was 12 hours post infection. The 14th GPV-CZ-ca virus titer of TCID50 was about 10-9.4/0.2ml. Compared with field virus, GPV-CZ-ca showed no lethality in goose, but field virus could cause 100-percentage mortality. The results demonstrated that GPV-CZ-ca has been attenuated gradually by passage in GEF. Goslings inoculated with 109TCID50 of GPV-CZ-ca could produce neutralizing antibody, the neutralizing antibody was achieved peak 22 days post inoculation. This virus can be used as candidate virus strain for the vaccine development in future.A pair of the primers based on the sequence in Genbank was designed to amplify the VP3 gene of GPV-CZ. The target fragment was inserted into pGEX-6P-1 vector, and then transformed into BL21 competent cells. The results demonstrated that the protein could be expressed in E. coli in high efficiency. Have optimized the expression condition, the recombinant protein could express in high efficiency at different IPTG concentration after 5 hours induction. The recombinant protein expressed was about 84 kDa in western blot with polyclonal antibodies from mice immunized with concentrated GPV. These results indicated that the VP3 expressed in vitro is very useful for diagnosis of goose plague.