Co-expression of the major immunogenic genes of goose paramyxovirus and goose parvovirus in Bac-to-Bac baculovirus expression system and immunogenicity of the recombinant baculovirus

According to the nucleotide sequence of Goose paramyxovirus(GPMV)NA1 strain,a pair of primers were designed and synthesized,to take the pGF plasmid as the template,the F gene fragment was amplified by PCR,with the stop codon to be knocked out.Then the F gene fragment was insered into the transposon vector pFastBacⅠ,constructed the pFastBac-F recombinant plasmid.According to the VP3 gene nucleotide sequence of goose parvovirus(GPV)GD-01 strain,a pair of primers with a Linker were designed and synthesized,the VP3 gene fragment obtained by PCR,and named LVP3.A recombinant transposon vector(pFF-LVP3) was contructed by inserting the LVP3 gene fragment into the pFastBac-F recombinant plasmid.Then pFF-LVP3 was transformed into E.coli DH10Bac,which contains bacmid and helper plasmid,and transposition was carried out,recombinant bacmid was constructed in it.The recombinant bacmid was transfected into Sf9 cells to generate recombinant baculovirus expressing F-VP3 recombination protein.The results of SDS-PAGE and Western-blot showed that the molecular weight of the expressed F-VP3 recombinant protein was about 123 000,which was recognized specifically by polyclonal antibody against GPMV and GPV.Based on the results of Western-blot,the goslings were immunized with production of expression of the recombinant baculovirus in Sf9 by intramuscular injection.After three times of immunization,the specific antibodies against GPMV and GPV were determined by hemagglutination inhibition(HI)and neutralization test.The results indicated that the F-VP3 recombinant protein expressed by recombinant baculovirus could induce GPMV HI antibody and GPV neutralizing antibody titer.This study laid the foundation for development of the subunit vaccine,recombinant live viral vector vaccine of GPMV and GPV.