Immunogenicity Differences Between The Proteins PrM/E And C Of Duck Tembusu Virus By Delivering Of Attenuated Salmonella

In this study,SL7207(PVAX1-SME)and SL7207(PVAX1-C)were two vaccine strains that constructed by our laboratory,the plasmids carrying PrM/E and C gene of DTMUV were transfected into attenuated Salmonella to obtain the two vaccine strains.We detected the levels of cytokines and distribution of vaccine antigens of ducklings after immunization,and analyzed the immunogenicity differences between the proteins PrM/E and C.The main contents of the study are as follows: 1.The difference of immune effect between protein PrM/E and C of Duck Tembusu delivered by attenuated SalmonellaTo determine the immune dose of the vaccine,we work out their living bacteria concentration under certain conditions by viable count meth;and the Duck Tembusu virus inactivated vaccine was prepared by optimization scheme.72 ducklings were divided into 4 groups on average and immunized with inactivated vaccine(group A),SL7207(PVAX1-SME)(group B),SL7207(PVAX1-C)(group C)and PBS(group D)at 8th and 16 th day,we collected serum at 16 th,24th,32 nd,40th,48 th and 56 th day.The antibody level and neutralization ability of ducks after immunized were detected by ELISA and serum neutralization test.The results showed that the neutralization ability of group B was higher(P<0.05)than that in group C from 16 th to 40 th day,and at other time,there was no difference between the two groups.The antibody level of group B was higher than(P<0.05)that of group C from 24 th to 40 th day and at 56 th day,there was no difference between the two groups at other time.The results showed that the PrM/E protein had a better immune effects.2.The difference between PrM/E and C protein stimulated by attenuated Salmonella in cytokine mRNAThe expression of the IL-4,IL-6 and IFN-gamma of duck immunized at 16 th,24th,32 nd,40th,48 th and 56 th day was analyzed,the results indicated that the expression of IL-4 in group B raised from 24 th to 56 th day(P<0.05),the expression of IL-4 in group C raised from 24 th to 48 th day(P<0.05),but the expression of IL-4 in group B was higher than that in group C(P<0.05)after 24 th day.The expression of IL-6 in groups B and C both began to increase significantly(P<0.05)from 16 th to 56 th day,respectively,but the expression of IL-6 in group B was higher(P<0.05)than that in group C at all other times except for 56 th day.The expression of IFN-? in group B raised(P<0.05)from 16 th to 48 th day,which only increased(P<0.05)at 16 th day in group C.That showed that these two proteins were more likely to stimulate the increase of IL-4 and IL-6 expression,but the changes in the group B were greater and longer than that in group C.3.Analysis of the distribution of vaccine antigen in ducks by immunohistochemical stainingThe immunohistochemical staining was used to find out the distribution of vaccine antigen in ducks.The distribution of the proteins in the intestines was similar in two groups,but there was a big difference in the essential organs.In the heart,the proteins of group B and C were detected strongly(+++)at 24 th day,but the protein of group B began to decrease at 56 th day and the protein of group C decreased at 40 th day;in the liver,the protein of group B was distributed strongly(+++)from 16 th to the last day,the protein of group C was detected strongly(+++)at 32 nd day and decreased at 48 th day;in the spleen,the protein was detected strongly(+++)at 24 th day in group B and at 32 nd day in group C,but both of two groups decreased at 56 th day;the two groups showed similar changes in the lung,which were strong(+++)at 24 th day and weakened at 40 th day;in kidney,the two groups were detected strongly(+++)at 24 th day,and the group B decreased at 56 th day and the group C decreased at 48 th day.That meant the existence time of protein in group B was longer than that in group C.In the end,compared to C protein of DTMUV,PrM/E protein has a longer existence in ducks,and its stimulation of cytokines was more significant.Thus,the immune effect of PrM/E protein as a vaccine antigen was better than that of C protein,which made the PrM/E protein has a better immunogenicity.