Mycobacterium Avium Subsp. Paratuberculosis Hsp65 And Goose Parvovirus VP3 Gene Construction Of Eukaryotic Expression Vector And Immune Effect Study

Gosling Plague also called goose parvovirus infection,it is caused by goose parvovirus,acute or subacute septic infectious diseases 4-20 day age goose and Muscovy ducks is infected very easy.With acute enteritis, liver, kidneys and heart essence organs pathological changes.its spread very quickly,morbidity and mortality rate very high.At present,Although single-gene vaccine can receive efficient expression outside of animal body,but elicited very weak immune response in animal body,this is limits the gene vaccine practical application.One of astrategies is join the immune adjuvants to enhance gene vaccine immune effect.As mycobacteria advantage antigen, Hsp65 not only can induce or enhance humoral immune responses and cellular immune responses,but also has carrier effect and molecular adjuvants function.The purpose of this research is to explore Mycobacterium avium subsp Paratuberculosis Hsp65 to GPV VP3 gene vaccine adjuvants role, take MAP hsp65 gene and GPV VP3 gene cloning to eukaryotic expression vector pVAX1,by PCR,Restriction Enzyme and Sequence Analysis,Proof that we obtained right recombinant plasmid pVAX1-hsp65-VP3,The transfection of this plasmid to Vero cell by liposome method. Confirmed that the recombinant plasmid pVAX1-hsp65-VP3 can transient expression in Vero cell by RT-PCR and indirect immunofluorescence,With respectively and fractionated,take pVAX1-hsp65-VP3 and pVAX1-VP3 to goose 100μg per one by intramuscular injection,Detection T activity by lymphocyte transformation testdetection specific antibody level in serum by indirect ELISA,the results shows that pVAX1-hsp65-VP3 T lymphocytes proliferation index to pVAX1-VP3 and attenuated vaccine difference was significant(P<0.05) at 21 days and 28days, Indirect ELISA results shows that pVAXl-hsp65-VP3 immunized group to pVAX1-VP3 and attenuated vaccine immunized group GPV specific antibody level in goose serum,difference was significant(P<0.05); Safety test results shows that the goose no abnormalities by eye and anatomy inspection.The above test illustrate that MAP Hsp65 enhance immune effect of pVAXl-VP3,Establish the foundation for gosling plague gene vaccine and Hsp65 adjuvants role.