Immune Effect Of Eukaryotic Expression Plasmid With Goose Parvovirus (GPV) VP3 Gene In Mice

Gosling Plague was a kind of acute or subac septico-infectious disease that was caused by Goose Parvovirus.Its characteristic affections were exsudation enteritis,the mucosa ecderon of intestina parva had necrosis and exfoliation,intestinal tract embolism.This disease mainly infected 4-20 days old gosling and savages ducks which were out of their shell.Related study on the gosling plague was developed in the world and had got a certain achievement after the goose parvovirus(GPV) was isolated from the goose with goose embryo by Fang Ding-Yi in China in 1956.In our country breeding googse business has a good tendency in development,but once Gosling Plague happen that will bring about huge economic losing to breeding googse specialized households.This because Gosling Plague disease has fast speed in infection and a high mortality which is reach up to 90%-100%.Traditional vaccine have a certain active effection on preventing and controlling to the disease,but their own have some inevitable disadvantages.For instance,inactivated vaccine lost some important epitopes so it had a bad immunogenicity,live vaccine easily happened virulence atavism to interfere with quarantining,and so on.To compare with these traditional vaccine,DNA vaccine has below advantages: stably expressing in vivo,and having time long immunization;getting out of pathogenic microorganism,so having free from infection potential danger;Requiring low dose,so making an effect as long as ng level.The gene VP3 of GPV is the main protective antigen gene,and its codogenic protein is main constituent of viral nucleocapsid,about 80%in protein level.The protein which exposed the surface of virion can induce organism to produce neutralization antibody.Making researcher on the immune effect of VP3 gene eukaryotic expression plasmid to lay a foundation for goose plaque DNA vaccine.In our experiment empty vector plasmid pVAX I and Eukaryotic expression plasmid with goose parvovirus(GPV) VP3 gene(pVAX I/VP3) which was successfully constructed by our laboratory and transferred to Vero cells with high efficiency were both extracted abundantly by alkaline lysis method.Ten 6 weeks old female BALB/c mice were immunized i.m.with eukaryotic expression plasmid pVAX I/VP3,empty vector plasmid pVAX I and normal saline at the two points respectively,totally immunized four times.In asepsis condition,the spleen of the immunized mice were took out,and then lymphocyte were isolated. The T lymphocyte proliferation activity were detected by lymphocyte conversion test endocellular enzyme MTT(3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) based colorimetic assay with the stimulation of GPV specific antigen,and the T lymphocyte population of CD3~+,CD4~+,CD8~+were detected by flow cytometry.After using statistic software SPSS 11.5 edition,the results of pVAX I/VP3 experiment group,pVAX I control group and normal saline control group all didn't reach to the statistical significance level on both T lymphocyte proliferation activity and cell population.All these showed that the eukaryotic expression plasmid pVAX I/VP3 which was successfully constructed by our laboratory could induce mice to make a low cellular immunity level.This was the similar with the reportorial finding from Zhang Zhongyang, et.al.But the reason of the finding was still not understand,presuming that maybe relate to the physiology condition of the immunized mice,the many influencing factors of lymphocyte conversion test,and so on.The serum of the immunized mice was separated,and GPV antigen were purified with according to the method described in the paper of Li Maoxiang,2.2%of NaCl and 6%of PEG were chosen as the conditions to sediment most of the virus,they were then purified through the Sepharose-4B column chromatography. The double square matrix method was used to decide the best working concentration of the purified GPV antigen was 10μg/mL and the best working dilution of the pooled serum of mice which were immunized by eukaryotic expression plasmid pVAX I/VP3 was 1:600,and then on this basis the indirect ELISA method by which the GPV specific antibody was detected was set up.To utilize the indirect ELISA method mentioned above,the level of the GPV specific antibody from the immunized mice was detected.After using statistic software SPSS 11.5 edition,the GPV specific antibody level of mice immunized with pVAX I/VP3 plasmid proved significantly comparing with those of mice in empty vector control group and negative control group,but no significant difference between the pVAX I control group and normal saline control group.Accordingly,this eukaryotic expression plasmid pVAX I/VP3 could induce immunized mice to make apparent humoral immunity.Our experiment showed that the eukaryotic expression plasmid pVAX I/VP3 which was successfully constructed by our laboratory could induce the immunized mice to make the GPV specific antibody,and laid a foundation for researching Gosling Plague DNA vaccine,although didn't make the ideal level of cell immunity.