Construction Of Goose Parvovirus VP2 Gene And Goose IL-2 Fusion Gene

Goose Plague(GP),caused by Goose parvovirus(GPV),is a fast transmission,high mortality and high contagious disease.The morbidity and mortality of 5 to 13-day goose may be up to 100%.At present,the weak poisonous vaccine and the oil emulsion deactivation vaccine are used for preventing and controlling the disease.The price of the weak poisonous vaccine is low and the effect is fine,but it is easy to cause the viral dissemination.The deactivation vaccine is safe and produces good humoral immunity reaction,but the deficiency is that the dosage is large and it can not induce organism to produc cellular immunity.A series of researance of nucleic acid vaccine has brought the new hope for the immunoprophylaxis of GP,but some researches showed that nucleic acid vaccine also has some deficiencies.Therefore,how to strengthen the immunity effect of the nucleic acid vaccine become the key point of vaccine research.With the deeply research of the cell factor such as IL-2,people discovered that it has the obvious molecular adjuvant effect which can enhance the antigen immunogenicity or enhance the antigenic reactivity of the organism.1.Construction of DNA vaccine pCDNA3.1(+)-VP2In this study,according to the GPV-B strain genomic sequence accessed in GeneBank,a pair of specific primers were designed.The BamHⅠsite and XhoⅠsite were appended to the upstream and downstream of primers in order to express easily.DNA were extracted from the culture of Goose parvovirus,and then the gene fragment was amplified by PCR.The PCR product was ligated to Vector of pMD18-T,then the recombinant plasmid was transformed into Escherichia coli(E.coli) DH5a.Positive clone with interest gene was indentificated by restriction enzyme analysis,PCR and DNA sequencing.The target gene was subcloned into expression vector pCDNA3.1(+),and the recombinant plasmid pCDNA3.1(+)-VP2 were constructed.2.Construction of DNA vaccine of pCDNA3.1(+)-VP2-IL-2 According to the GPV-B strain and Goose Interleukin-2 genomic sequence accessed in GeneBank,a pair of specific primers were designed respectively.The BamHⅠsite and SalⅠsite were added to the upstream and downstream of GPV VP2 gene primer respectively.SalⅠsite were appended to the upstream of IL-2 gene primer,and XhoⅠsite,HindⅢsite were added to its downstream.Goose Interleukin-2 were amplifed by RT-PCR,and then cloned to pMD18-T.The DNA of GPV were used as the template,and VP2 were amplified by PCR,then the PCR production were ligated to the recombinant plasmid containing IL-2 gene.The VP2 gene did not contain the termination codon,the IL-2 gene does not contain the outset codon,so the heads and tails of the two gene were connected.At last,the recombinant plasmid DNA vaccine of pCDNA3.1(+)-VP2-IL-2 were formed by ligation of the fusion gene VP2-IL-2 and the expression vector pCDNA3.1(+).3.White Mice Immunnity ExperimentIn the White Mice Immunnity experiment,the mice were divided into five groups,and there were 8 mice in every group.The groupⅠ,Ⅱ,Ⅲ,ⅣandⅤwere immunized with the DNA vaccine of pCDNA3.1(+)-VP2-IL-2,the DNA vaccine pCDNA3.1(+)-VP2,the GPV attenuated vaccine,the pCDNA3.1(+) vector and sterile physiologic saline respectively.There were 14 days between the first and second immunization,and after two weeks the third immunization were carrried out.The immune effect of the DNA vaccine were determined by indirect ELISA.The result indicated thatⅠgroup andⅡgroup could induce the organism to produce cellunar immunity.After the first immunization,the content of the IL-4 and INF-γofⅠandⅡgroup mice was significantly higher thanⅢgroup(P＜0.01),the that ofⅢgroup was higher thanⅣand Vgroup(P＜0.01).After the second immunization,the content of the IL-4 and INF-γofⅠgroup mice was very significant higer thanⅡgroup(P＜0.01).