Linear B-Cell Epitope Mapping For Non-Structural And Structural Proteins Of Goose Parvovirus

Goose parvovirus(GPV) infection,also known as goose plaque or Derzsy's disease,causes high mortality in domestic goslings(Anser anser vardom) and Muscovy ducklings(Cairina moschata).It caused 90 to 100%mortality in susceptible goslings under the age of 4 days to 20 days.GPV infection has been reported from many goose-producing countries,and caused huge economic loss.Owing to the hindrance to development of goose cultural industry because of this desease,there needs accurate,convenient diagnostic method for early diagnosis avoiding the interference of maternal antibodies.Meanwhile,it needs monitor the level of maternal antibodies. Furthermore,it needs analyze the viral antigen to select the suitable peptides be as subunits vaccine for desease prevention.Notwithstanding many veterinary researchers paid close attention to GPV,the study about the antigenicity of viral protein fell behind other aspects.The genome of the members of parvovirinae is small and the capsid structure is simple,so it is easy to eliminate independent antigenic component.Thus,it has superiority in prepareing subunit vaccine and recombinant diagnostic antigen.Except for this,GPV not only has the high mortality feature of autonomously replicating parvoviruses,but also has the genetic feature of dependovirus.Therefore,we can study the GPV collateing the research finding of other parvovirus.In this study,two recombinant plasmids harboring non-structural and structural protein gene named pMD18-T-NS1 and pMD18-T-NS1 were constructed and sequenced.The two recombinant plasmids were used as the templates for PCR in the next procedure.To map the antigenic epitopes of non-structural protein,a set of partially overlapping fragments of 50-60aa spanning the protein were designed.The totals of 15 pairs of primers were synthesized. All the forward primers contained a BamHI restriction site,and reverse primers contained a termination codon and an XhoI restriction site.PCR products were cloned into expression vector, pGEX-6P-1(Amersham Pharmacia Biotech,Sweden) and the map of these fragments confirmed by sequencing.Each fragment was expressed as glutathione S-transferase(GST) fusion proteins in Escherichia coli Rosetta(DE3)pLysS and purified by elution from sodium dodecyl sulphate(SDS) polyacrylamide gels.Anti-GST tag MAb was used to identify the purified protein.Then western blot reactivity of these short peptide fused protein to viral infected sera were surveyed.Linear immunodominant B-cell epitopes were primarily found in four fragments:NS(453-514), NS(485-542),NS(533-598),NS(575-627).The further 7 overlapping fragments of the NS1 protein from 453 to 627aa were also expressed in E.coli.The further results of mapping NS1 protein immunodominant epitopes are fragments NS(498-532) which showed strongly positive,fragments NS(485-514),NS(523-556),NS(543-573),NS(564-598) and NS(599-627) which were weakly positive.To map the antigenic epitopes of structural protein,a set of partially overlapping fragments of 50-60aa spanning the protein were designed.The totals of 19 pairs of primers were synthesized. Western blot reactivity of these short peptide fused protein to viral infected sera were surveyed. Linear immunodominant B-cell epitopes were primarily found in fragments:VP(35-100), VP(81-136),VP(124-161),VP(146-198),VP(423-491),VP(531-595),VP(616-669) and VP(678-732).The further 16 overlapping fragments of the VP1 antigenic protein were also expressed in E.coli.The further results of mapping VP1 antigenic protein immunodominant epitopes are fragments:VP(35-63),VP(50-81),VP(111-145),VP(136-161),VP(423-453), VP(462-491),VP(531-560),VP(548-577),VP(616-647),VP(634-669),VP(678-706) and VP(697-732).Thus,the non-structural protein linear B-cell epitopes are located on the C-terminal, (485-627aa).The structural protein linear B-cell epitopes are located on the 35-81aa,111-198aa, 423-453aa,462-491aa,531-577aa,616-669aa and 678-732aa.After goose parvovirus infection,the host principal immune reaction is humoral immune reaction.Identification of B-cell epitopes can reveal the essence of humoral immune reaction and supply the blank in GPV research.Meanwhile,because of GPV supposed to be the antecedent of autonomous parvovirus and dependovirus,the study about GPV non-structural and structural protein antigenicity can complete the knowledge of interaction between parvovirus and their hosts. In a word,identification of antigenic epitopes of the non-structural and structural protein of GPV may be helpful in understanding molecular properties of non-structural and structural protein of GPV,as well as relationship between immunogenicity or pathogenesis and gene variations of the virus.Therefore,the results from the study may also be useful for diagnosis of GPV infection based on antigenic epitope or developing a new strategy for vaccine design.