Immune Effect Of DNA Of Eukaryotic Expression Vector Of Goose Paramyxovirus HN Gene In Mice

A new viral disease caused by goose paramyxovirus(GPMV) is an acute and violent infectious disease for geese,which characterized high mortality and result to a serious disserve in goose breeding industry.At present,there is not specific medicine to control GPMV in clinic.However,traditional live vaccine could not produce enough protective immunity and inactive vaccine have the disadvantage of virulence reversion.With the development of molecular biology,how to prevent and control the disease with genetic engineering vaccine becomes an urgent problem.According to studies on GPMV,the enveloped virus has a negative-sense,single-stranded RNA genome which codes for six proteins including nucleoprotein(NP),phosphoprotein(P),matrix(M) protein, RNA-directed RNA polymerase(L),hemagglutinin-neuraminidase(HN) protein and fusion(F) protein. Hemagglutinin-neuraminidase(HN) protein is one of the major protective antigen of GPMV which including hemagglutinin activity(HA) and neuraminidase activity(NA),HA is responsible for identifying the sialic acid-containing receptors and taking part in the effect of the viral attachment to target membranes,NA displays receptor-destroying activity and virosome-releasing activity,HA and NA play essential roles on the infection of virus.Therefore,it is significant to construct an eucaryon expression vector with major protective antigen gene HN as DNA vaccine to prevent the disease.This research is based on the study of partial biological characters of GPMV jilin strain.The empty vector plasmid pVAXâ… and eukaryotic expression plasmid of goose paramyxovirus HN gene(pVAXⅠ—HN) which was constructed by our laboratory successfully were both extracted abundantly by alkaline lysis method. BALB/c mice were injected i.m with the largely extracted pVAXⅠ—HN plasmid and empty vector pVAXâ… to observe the immune effect.30 BALB/c mice were randomly divided into three groups,each group 10 mice,which were named respectively pVAXⅠ—HN plasmid test group,empty vector pVAXâ… control group and normal saline negative control group.Mice were killed after immunized four times.The spleens of the immunized mice were took out and isolated in the asepsis condition.The T lymphocyte proliferation activity was detected by MTT,the T lymphocyte population of CD3⁺,CD4⁺,CD8⁺ were detected by flow cytometry.The serum of the immunized mice was separated,and then the double square matrix method was used to establish the best working concentration of indirect ELISA to detect the serum antibody against GPMV.MTT test showed no significant difference between the mice in the test group and the two control groups, the statistical results of the stimulation index(SI) respectively were 1.18±0.03,1.12±0.04 and 1.16±0.03. Flow cytometry test also showed no significant difference between the mice in the test group and the two control groups about the T lymphocyte population of CD3⁺,CD4⁺,CD8⁺,in which,the statistical results of CD3⁺ T cells respectively were 40.66%±1.43%,41.07%±4.72%and 40.94%±5.32%,the statistical results of CD4⁺ T cells respectively were 26.00%±1.83%,25.32%±2.89%and 25.77%±4.01%,the statistical results of CD8⁺ T cells respectively were 12.55%±1.19%,12.79%±2.08%and 12.42%±1.87%.The best working concentration of indirect ELISA used the double square matrix method were established:antigen concentration was 30μg/mL,serum dilution was 1:400,concentration optimization of HRP conjugated antibody against mice IgG was 1:5000.The OD₄₉₂ value of each group mice were determined on the basis of the best working concentration above,the results showed that the specific humoral immune response of the mice in the test group was increased significantly compared to mice in the empty vector control group and normal saline negative control group.The OD₄₉₂ value of ELISA were respectively 1.36±0.10,0.36±0.02 and 0.31±0.02.Therefore,the results showed that the constructed eukaryotic expression plamid of GPMV with HN gene didn't make the ideal level of cell immunity,but it induced specific humoral immunity and laid a foundation for further researching goose paramyxovirus DNA vaccine.