| According to the published interleukin-2(IL-2) gene sequence of Sichuan White Goose from GeneBank,PCR primers were designed by OLIGO6.0,the gene of goose IL-2 mature fragment was obtained by PCR,the sequence was analyzed by bioinformatics. Then the following researches were done:GoIL-2 gene expression vector construction, expression product purification and its biological activity detection,and its effect of adjuvant to gosling plague attenuated vaccine and gosling plague VP3 genetic engineering subunit vaccine in young geese and googe.The results were as follow:1 GolL-2 prokaryotic expression,product purification and bioactivityThe fragment of GolL-2 maturation protein gene was obtained by PCR and cloned into pMD18-T vector.And it was subcloned into pET32a+ to construct recombinant expression plasmid.Then it was transformed into Escherichia coli BL21(DE3) and expressed after induced by IPTG.The molecular weight of recombinant was 34KD,and the expression products were soluble recombinant proteins which was 42.5%of total protein in BL21(DE3).The recombinant protein was purified by metal immobilization affinity chromatography(Ni+-MIAC).The recombinant protein was refolded by the methods of dialyze,then the bioactivity of the recombinant protein was detected by MTT,and its bioactivity was 110672U/mg.2 Recombinant GoIL-2 effects of adjuvant to gosling plague VP3 genetic engineering subunit vaccine and GPV attenuated vaccine in young geeseRecombinant GoIL-2 was administrated to 28-day old geese with 16 antigenic determinants of gosling plague virus VP3 genetic engineering subunit vaccine(Ag16v),10 antigenic determinants of gosling plague virus VP3 genetic engineering subunit vaccine(Ag10v),GPV attenuated vaccine, respectively,divided into 12 groups:500 U+Ag16v,1000U+Ag16v,2000U+Ag16v,500U+Ag10v, 1000U+Ag10v,2000U+Ag10v,1000U+GPV.Ag16v,Ag10v,GPV attenuated vaccine;rGoIL-2 and PBS were controls.(1)Cellular immunity:Peripheral blood samples were collected on 3d,7d,14d,21d,28d,35d and 49d after immunized.T-lymphocytes of Test groups were significantly(P＜0.01) or(P＜0.05) higher than PBS control group on 7-49d after immunized.①2000U+Ag16v were significantly different (P＜0.01) or different(P＜0.05)than Ag16v and 500U+Ag16v on 14-49d after immunized,significantly (P＜0.05) higher than the 1000U+Ag16v group on 21-28d.500U+Ag16v and Ag16v were not significantly different(P＞0.05).②2000U+Ag10v was significantly different(P＜0.01) or different (P＜0.05)than Ag10v and 500U+Ag10v on 14-49d after immunized,significantly(P＜0.05) higher than the 1000U+Ag10v group on 21-28d.500U+Ag10v and Ag10v were not significantly different(P＞0.05).③1000U+GPV were significantly different(P＜0.01) or different(P＜0.05) than GPV control group on 14-28d after immunized.GoIL-2 enhanced cellular immune responses which gosling plague VP3 genetic engineering subunit vaccine and GPV attenuated vaccine induced,and was some dose-dependent.(2)Humoral immunity:peripheral blood samples were collected on 3d,7d,14d,21d,28d,35d,49d,63d,77d,91d,105d,119d,133d,147d,161d and 175d after immunized.①ELISA antibody: Test group was significantly(P＜0.01) higher than PBS control group on 7-175d after immunized. 2000U+Ag16v was significantly different(P＜0.01) or different(P＜0.05) than Ag16v and 500U+Ag16v on 14-147d after immunized,significantly different(P＜0.01) or different(P＜0.05) than 1000 U+Ag16v on 21-28d after immunized.500U+Ag16v group and Ag16v were not significantly different(P＞0.05). 2000U+Ag10v was significantly different(P＜0.01) or different(P＜0.05) than Ag10v and 500U+Ag10v on 14-147d after immunized,significantly different(P＜0.01) or different(P＜0.05) than 1000U+Ag10v on 21d,49d,77d after immunized.500U+Ag10v group and Ag10v were not significantly different(P＞0.05).1000U+GPV group were significantly different(P＜0.01) or different(P＜0.05) than GPV attenuated vaccine on 14-147d.②Neutratizating antibody:Test group and the control group(except PBS,IL-2 control group) had a specific antibody GPV.2000U+Ag16v was significantly(P＜0.01) or significant(P＜0.05) higher than 500U+Ag16v group and Ag16v on 21-147d,significantly different (P＜0.01) or different(P＜0.05) than 1000U+Ag16v on 21-35d.500U+Ag16v group and Ag16v were not significantly different(P＞0.05).2000U+Ag10v were significantly different(P＜0.01) or different (P＜0.05) than Agl0v and 500U+Ag10v group on 14-147d,significantly different(P＜0.01) or different (P＜0.05) than 1000U+Ag10v on 28d.500U+Ag10v group and Ag10v were not significantly different (P＞0.05).1000U+GPV was significantly different(P＜0.01) or different(P＜0.05) than GPV attenuated vaccine on 21-147d.Humoral immune tests showed that the rGoIL-2 enhanced humoral immune responses which gosling plague VP3 genetic engineering subunit vaccine and GPV attenuated vaccine induced,and was some dose-dependent.3 Recombinant GoIL-2 effects of adjuvant to gosling plague VP3 genetic engineering subunit vaccine and GPV attenuated vaccine in breeding geeseRecombinant GoIL-2 was administrated to geese with Ag16v,Ag10v and GPV attenuated vaccine respectively.Ag16v,Ag10v,GPV attenuated vaccine,IL-2 and PBS were controls.Peripheral blood samples were collected on 3d,7d,14d,21d,28d,35d,49d,63d,77d,91d,105d,119d,133d,147d,161d and 175d after immunized.Each groups hatched eggs,and the future generations of gosling were used to infection protection test with 100 LD50 GPV-CHv strong strain.Results:(1) Serum antibody ELISA:6000 U+Ag16v,6000U+Ag10v and 6000 U+GPV,7-147 d were significantly different(P＜0.01) or different(P＜0.05) than Ag16v,Ag10v and GPV control group,respectively.(2) Serum Neutratizating antibody:6000 U+Ag16v,6000U+Ag10v and 6000 U+GPV,21-147 d were significantly different(P＜0.01) or different(P＜0.05) than Ag16v,Ag10v and GPV control group.(3)Yolk antibody ELISA:6000U+Ag16v,6000U+Ag16v and 6000U+GPV were significantly different(P＜0.01) or different(P＜0.05) than Ag16v,Ag10v and GPV respectively on 7-147d.(4) Yolk Neutratizating antibody:Test group and the control group(except PBS,IL-2) had a specific GPV antibody.6000U+Ag16v,6000U+Ag16v and 6000U+GPV were significantly different(P＜0.01) or different(P＜0.05) than Ag16v,Ag10v and GPV group on 21-147d aider immunized.GoIL-2 enhanced yolk antibody levels which gosling plague VP3 genetic engineering subunit vaccine and GPV attenuated vaccine induced.(5)Infection rate of protection:6000U+Ag16v,6000U+Ag10v and 6000U+GPV infection rate of protection were higher than Ag16v,Ag10v and GPV attenuated vaccine,respectively. Immunization period of protection was 140d,respectively.Ag16v,Ag10v,GPV attenuated control immune protection period were 119 d,119,133 d,respectively.Test group of its future generations of immune protection period was longer than the control group for 7-21 d.GoIL-2 improved the infection rate of protection of gosling plague VP3 genetic engineering subunit vaccine and GPV attenuated vaccine after their goslings and future generations. |
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