Construction Of Goose Parvovirus VP3 Gene Recombinant Adenovirus And Its Immunogenicity

Gosling Plague,seriously harming the healthy development of goose industry,was a kind of acute or sub-acute infection caused by goose parvovirus(GPV),and its mortality rate was up to 90% to 100% in 1-2-week-old goslings.GPV was an important member of the parvovirus genus and VP3 protein was the major structural protein composing virus capsid which could induce neutralizing antibodies.Thus,VP3 became the preferred protein for studying gene engineering vaccine of gosling plague.In this study,VP3 gene,amplified by PCR,was recombined to adenovirus vector,and then recombinant adenovirus efficiently expressing VP3 protein was packed in vitro,finally,immunogenicity was observed.This study was aiming at laying foundation for GPV recombinant live vector vaccine.(1)Construction of recombinant shuttle plasmid containing VP3 gene: VP3 gene with the length of 1605 bp was amplified by PCR using p DC-VP3 recombinant plasmid as template,and was inserted in the shuttle plasmid pac Ad5 CMV K-Np A,obtaining recombinant shuttle plasmid pac Ad-VP3.(2)Construction of recombinant adenovirus containing VP3 gene: pac Ad-VP3 and pac Ad5 9.2-100 backbone plasmid,linearized by Pac I,were co-transfected into HEK293 AD cells mediated by liposome,obtaining recombinant adenovirus rAd-VP3.The virus titer could be up to 10-8.23/0.1m L TCID50 when cultured to the 6th generation.(3)Identification of rAd-VP3: 1605 bp DNA fragment was amplified from rAd-VP3 by RT-PCR;specific protein strips of 57 k D was detected from HEK293 AD cells infected by rAd-VP3 with the method of Western blot;HEK293AD and GEF infected by rAd-VP3 appeared green fluorescence after stained with GPV antiserum and labeled goat anti-mouse Ig G.Results above showed that recombinant VP3 protein had specific immune response with GPV antiserum.(4)Immunogenicity of rAd-VP3: serum antibody titers of Kunming mouse(SPF)immuned with rAd-VP3 could reach 1:264,and cytopathetic effect of GEF caused by GPV was restrained by the mouse serum;high levels of the GPV antibodies in goslings were detected after being immuned 14 d,and kept high after 28 d.In summary,this study successfully constructed recombinant adenovirus rAd-VP3 expressing the major structural protein of VP3.rAd-VP3 could infect HEK293 AD and GEF,in which VP3 protein was highly expressed;rAd-VP3 had good immunogenicity and could induce neutralizing antibodies in mice and goslings.The study laid a good foundation for the study of safe and efficient GPV recombinant vaccine.