Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV S...Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV SYG-61 were isolated.The genes of scFv antibodies were derived from goslings immunized with GPV SYG-61,and scFvs were subcloned into a pBSD vector for the construction of pBSD-scFv libraries.The pBSD-scFv libraries were screened following three rounds using VP2(protective antigen of GPV)as the bait by flow cytometry(FCM).After screening,the 15 clones with high mean fluorescence intensity(MFI)were isolated and sequenced.These 15 scFvs were expressed by pET-28a(+)in E.coli.The specificity and affinity of the 15 purified scFvs were successfully confirmed by ELISA.In the preliminary neutralization experiment on primary goose embryo fibroblast(GEF)in vitro,three of the 15 purified scFvs(named scFv-10,scFv-11 and scFv-50)showed significant neutralizing capacities.The study generated the first goose-origin neutralizing scFv against GPV and laid the foundation for the appearance of full-length goose-origin neutralizing monoclonal antibody against GPV.展开更多
[Objective] The paper was to develop a subunit vaccine candidate for prevention and control of goose parvovirus infection. [Method]Based on the prokaryotic expression system, the antigenic epitopes and locations of th...[Objective] The paper was to develop a subunit vaccine candidate for prevention and control of goose parvovirus infection. [Method]Based on the prokaryotic expression system, the antigenic epitopes and locations of the structural protein VP3 were predicted by software analysis,and the region displaying a large portion of antigenic epitopes was amplified by PCR. The target VP3 DNA fragment was inserted into pET-30 a-VP3 vector, was transformed into Escherichia coli BL21 competent cells for protein expression and animal test. The SPF chickens were immunized with the recombinant protein and the antisera were collected for neutralization test by using a goose embryo fibroblast. [Result] The recombinant plasmid was constructed, and the target region of VP3 protein was expressed efficiently in a soluble form. The neutralizing titers of antisera could reach up to-2.608. [Conclusion] The target region displaying a large portion of antigenic epitopes of the structural protein VP3 could be expressed efficiently in soluble form, and the expressed protein could induce neutralizing antibodies in SFP chicken.展开更多
基金Supported by the National Key R&D Program of China(2017YFD0501102,20I7YFD050I103-03 and 2017YFD0501004)Science and Technology Department of Heilongjiang Province(GX18B018)Education Department ofHeilongjiang Province(TSTAU-R2018017)。
文摘Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV SYG-61 were isolated.The genes of scFv antibodies were derived from goslings immunized with GPV SYG-61,and scFvs were subcloned into a pBSD vector for the construction of pBSD-scFv libraries.The pBSD-scFv libraries were screened following three rounds using VP2(protective antigen of GPV)as the bait by flow cytometry(FCM).After screening,the 15 clones with high mean fluorescence intensity(MFI)were isolated and sequenced.These 15 scFvs were expressed by pET-28a(+)in E.coli.The specificity and affinity of the 15 purified scFvs were successfully confirmed by ELISA.In the preliminary neutralization experiment on primary goose embryo fibroblast(GEF)in vitro,three of the 15 purified scFvs(named scFv-10,scFv-11 and scFv-50)showed significant neutralizing capacities.The study generated the first goose-origin neutralizing scFv against GPV and laid the foundation for the appearance of full-length goose-origin neutralizing monoclonal antibody against GPV.
基金Supported by Youth Fund Project of Natural Science Foundation of Shandong Province(ZR2014CQ009)Science and Technology Development Program of Binzhou City(2013GG0304)
文摘[Objective] The paper was to develop a subunit vaccine candidate for prevention and control of goose parvovirus infection. [Method]Based on the prokaryotic expression system, the antigenic epitopes and locations of the structural protein VP3 were predicted by software analysis,and the region displaying a large portion of antigenic epitopes was amplified by PCR. The target VP3 DNA fragment was inserted into pET-30 a-VP3 vector, was transformed into Escherichia coli BL21 competent cells for protein expression and animal test. The SPF chickens were immunized with the recombinant protein and the antisera were collected for neutralization test by using a goose embryo fibroblast. [Result] The recombinant plasmid was constructed, and the target region of VP3 protein was expressed efficiently in a soluble form. The neutralizing titers of antisera could reach up to-2.608. [Conclusion] The target region displaying a large portion of antigenic epitopes of the structural protein VP3 could be expressed efficiently in soluble form, and the expressed protein could induce neutralizing antibodies in SFP chicken.
