Construction And Identification Of A Recombinant Adenovirus Expressing Of Vp3Gene Of Goose Parvovirus

VP3protein of nuclear capsid is the major structural proteins and protective antigen of goose parvovirus (GPV), it accounts for80%of the total protein of GPV, and has the high conservative, induces neutral antibody. In order to construction of a recombinant adenovirus expressing of VP3gene of goose parvovirus, this experiment mainly consists of two parts.1. Prokaryotic expression of VPS gene of goose parvovirus and preparation of its polyclonal antibody.This test in this experiment the saved goose parvovirus VP3gene recombinant plasmid cloning, on the basis of the construction of recombinant prokaryotic expression plasmid pET-30a-VP3, and in E.Coli BL21(DE3) express, to express the fusion protein SDS-PAGE and Western blot analysis appraisal, protein purification after rabbit immune experiment, the preparation of rabbit anti goose parvovirus mature VP3gene protein polyclonal antibody, measure the titer by indirect ELISA. Results show that we successfully construct recombinant prokaryotic expression vector of pET-3Qa-VP3, the expression of recombinant protein by SDS-PAGE and Western blot, size of molecular weight of about60Ku, and good reaction to VP3protein of GPV, The successful preparation of rabbit anti goose parvovirus mature VP3protein polyclonal antibody, titer of1:200indirect ELISA.2. Construction and identification of a recombinant adenovirus expressing of VP3gene of goose parvovirusIn order to study the goose parvovirus VP3gene recombinant live carrier vaccine inhibition of virus activity, this experiment using PCR amplification get goose parvovirus VP3gene, gene recombination technique was used to construct High-VP3transfer carrier, through the way of liposome mediated the recombinant plasmid transfection with Ad5QBI-HEK293cells, after being cells appear lesions (CPE), with the method of repeated freezing and thawing harvest recombinant adenovirus, restructure the PCR identification of virus VP3gene, and by using indirect immunofluorescence (IFA) test and Western blot test to test the recombinant virus, the antiviral activity of recombinant virus VP3gene analysis. Results show that the successful preparation of recombinant adenovirus rAd-VP3, the TCID50is10815/ml. PCR results showed that success will goose parvovirus VP3gene inserted into the adenovirus vectors, indirect IFA test and Western blot test results have shown that vp3gene expression successful in QBI-HEK293cells, and have good reaction to the original.In summary, the study had provided theoretical basis and the referenced value for the further experiments with recombinant adenovirus vaccine containing GPV-VP3in vivo.