Construction And Immunogenic Analysis On Suicidal DNA Vaccine Against Goose Parvovirus

Gosling Plague(GP)is a highly contagious and fatal disease infecting young geese and Muscovy,which is caused by Goose Parvovirus(GPV).GP is one of the most serious infectious disease hazard goose industry.Although the use of live attentuated vaccines could induce efficient protection,it is difficult to use as differentiating animals infected with vaccinated animals and GPV wild strain.Furthermore,it exists the possibility of virulence recovery in theory.Therefore,a safe,efficient and DIVA against PPRV infections is especially needed.VP3 protein is a glycoprotein of GPV that might induce a protective immune response.In our studies,a suicidal DNA vaccine expressing the VP3 protein of GPV was constructed.Moreover,the immunogenicity of the vaccines was evaluated in mouse and goslings.The primers used to amplify the VP3 gene was designed according to the GPV sequence in the GenBank.A pair of specific primers for amplifying the VP3 open reading frame was designed using Primer Premier software.DNA of GPV was extracted the allantoic fluid infected with GPV.The VP3 genes was amplified by PCR methods and then cloned to the pSCA1,the alphavirus replicon based vector.The resulting recombinant plasmids were identified with PCR,restrict enzymes and the sequencing,and named pSCA-VP3.After been transfected into the BHK-21 cells by Lipofectamine-2000,the results of IFA and Western blotting analysis showed that the recombinant plasmids could express the interest in vitro.Moreover,thirty 6-week BALB/c mice were randomly divided into three groups.The mice were injected intramusularly with pSCA-VP3 plasmid and empty plasmid pSCA1 thrice at 2-week intervals individually at the dosage of 100 ?g.The negative control group was injected with the same amount of PBS.0,2,4,6 weeks after the first immunization,peripheral blood serum of mice were collected and separated.Indirect ELISA were used for the detection of specific antibodys.Six weeks after immunization,the spleen of mouse was collected,and the lymphocyte proliferation test was performed to detect the level of celluar immune response.The results showed that pSCA-VP3 induced strong humoral and cellular immune responses.The levels of immune responses were significantly higher compared with pSCA1 empty vector and PBS negative control group.Finally,pSCA-VP3 recombinant plasmid and pSCA1 plasmid was injected intramuscularly into 7-day-old goslings,which has no maternal antibodies.Theimmunizations were conducted twice at 2-week intervals individually by 200 ?g.The negative control group was injected with the PBS.0,2,4 weeks after the first immunization of gosling collected peripheral blood serum was separated by an indirect ELISA detection of specific antibody levels.4 weeks after immunization simultaneous acquisition of anticoagulant gosling,lymphocyte proliferation test performed to detect the level of immune cells.At the same time,each group of goslings intraperitoneal injection GPV YZ strain which each by 100ELD50.The clinical symptoms and mortality rate of goslings was observe for seven days.The dead goslings were anatomied and the lesions of internal organs were observed.Moreover,HE staining and immunohisto chemistry analysis were used for pathological damage and the content of GPV in tissues.The goslings from pSCA-VP3 group and attenuated vaccine group induced both neutralizing antibodies and celluar immune responses.And the levels were significantly higher than empty vector group and negative control group.The results of protection test,HE staining and immunohisto chemistry showed that pathological lesions and virus content in pSCA-VP3 group and the attenuated vaccine group were better than pSCA1 empty vector and PBS control groups.In summary,the study constructed a suicidal DNA vaccine expressing VP3 gene of GPV.BALB/c mice immunized with pSCA-VP3 plasmid induced both GPV specific antibody and lymphocyte proliferative response.The results suggested that the recombinant plasmids could induce both humoral and cellular immune response against the GPV in miceThe pSCA-VP3 has a potential ability for preventing GPV affection.Importantly,immune effects detected of goslings also shown that the suicidal DNA vaccine pSCA-VP3 have better immune effect.The results of the studies provide a new thinking and options against GPV.It maybe provided useful technical support for further clinical research applications.