Construction Of Goose Parvovirus VP3 Gene Vaccines And The Preliminary Study On Their Immunogenicities

Goose parvovirus(GPV)infection is still a great threat for waterfowl industry in waterfowl breeding countries,its main prevention depends on the vaccination,but common vaccines usually have some defficincies,such as back to virulence or incomplete immune responses.Gene vaccine meets the future of vaccine development and could induce safe,low cost and complete immune responses in animals.This study took GPV VP3 gene as target gene for DNA vaccine construction,taken goose interleukin-2(gIL-2)and CpG motifs as adjuvant to improve the immunogenicity of GPV VP3 gene vaccines.1.In order to study the feasibility of VP3 gene vaccine,we firstly cloned VP3 gene using PCR,and then cloned the VP3 gene into pMD 18T-Simple T vector.VP3 gene was analylized its molecular character and antigenicity after sequencing,then subcloned into pcDNA3.1(+)for construction of pcDNA-VP3,it coube be expressed in COS-7 at 48h post transfection using indirect immunofluorescence antibody test.2.Using splicing by overlap extention PCR(SOE-PCR),gIL2-VP3 fusion gene was acquired,which lacked TAA stop codon of gIL2 gene and ATG iniation codon of VP3 gene,the two genes were linked by high hydrophilia amino acid(Gly-Gly-Gly-Ser) encoding nucleotides,gIL2-VP3 gene was then inserted into pcDNA3.1(+)under the control of CMV,pcDNA-gIL2-VP3 was constructed,which could be expressed in COS-7 cells 48h post transfection and its expression protein had bioactivity of IL-2(31.57U/ml).3.CpG ODN had been systhesized and ligated into pMD18T-Simple,then subcloned to pcDNA-gIL2-VP3,thus pcDNA-gIL2-VP3/CpG was constructed,which could stimulate the proliferation of PBMC in goose,the stimulation index was 3.06.4.28-day-old geese were immunized with three kinds of VP3 gene vaccine with different doses of 50μg,100μg and 200μg,anticoagulated blood was prepared and used for lymphocytes proliferation assay at 3d,7d,14d,21d,28d,35d and 49d post immunizaiton,the results indicated that,at 28d post immunization,pcDNA-gIL2-VP3 and pcDNA-gIL2-VP3/CpG had the highest values,stimulation index(SI)of pcDNA-gIL2-VP3/CpG immunized with 200μg doses was significantly different with pcDNA-gIL2-VP3 and pcDNA-VP3(P＜0.05).VP3 gene vaccine with adjuvant showed earlier peak of T lymphocytes proliferation abilities than pcDNA-VP3.5.Serum samples were collected from immunized geese at 3,7,14,21,28,35,49,63, 77,105,133,161,189,217 days post immunization,and indirect ELISA was used to detect the kinetics of IgG,the results indicated that,pcDNA-gIL2-VP3 and pcDNA-gIL2-VP3/CpG induced superior humoral immunity than pcDNA-VP3,200μg of pcDNA-gIL2-VP3/CpG induced the highest level of IgG,which was significantly different from pcDNA-VP3(P＜0.05)and other control groups(P＜0.01),VP3 gene vaccines possessed adjuvant induced better and longer immune responses than pcDNA-VP3 and GPV live attenuated vaccine group.6.28-day-old geese were immunized with 200μg each kind of gene vaccine,sera were collected for virus neutralization test at 15d,30d,45d,60d and 75d post immunization, the results indicated that,low levels of neutralization antibodies could be detected at 15d post immunization,then the neutralizaiotn in each gene vaccine group climbed slowly, pcDNA-gIL2-VP3 and pcDNA-gIL2-VP3/CpG peaked at 60d post immunization with titers of 1:178.5 and 1:198.2,there was no significant difference between them,but they were significantly different with pcDNA-VP3 group and GPV live attenuated vaccine group(P＜0.05).7.Three kinds of GPV VP3 gene vaccine were delivered into maternao geese with doses of 200μg each vaccine,yolk antibody and yolk neutralizing antibody were tested at 7d,14d,28d,42d,56d and 70d post immunization,geese eggs collected at 14d,28d and 42d post immunization were hatched for goslings used for virus protection assay.The results indicated that,yolk antibodies and yolk neutralizing antibodies at different times post immunization showed some correlations with the gosling virus protection test, pcDNA-gIL2-VP3/CpG was superior in the three gene vaccines and had the similar results with GPV-live attenuated vaccine.