Effect Of Different Molecular Adjuvant On EmIMP1 Immunization

The purpose of this experiment is to study the effect of molecular adjuvant FliC,CD40L and EDA on chicken Eimeria maxima protein EmIMP1 immunization.Firstly,we clone the target fragment,construct the expression vector pET-28a-EmIMP1,pET-28a-FliC-EmIMP1,pET-28a-EmIMP1-CD40L,pET-28a-FliC-EmIMP1-CD40L,pET-28a-EDA-EmIMPl.Then the expression vectors were transformed into E.coli and the recombinant proteins were purified.SDS-PAGE and Western-blot showed that protein EmIMP1 is about 60kDa;FliC-EmIMP1 is about 90kDa;EmIMP1-CD40L is about 90kDa;EDA-EmIMP1 is about 70kDa;and FliC-EmIMP1-CD40L is about 120 kDa.Finally,two-week-old male hyline brown chicken were immunized with the purified proteins.Two weeks later,the boost immunization was performed,after two weeks,chickens were infected with Eimeria maxima oocysts.The effect of adjuvant was evaluated from average body weight gain,oocyst output,intestinal lesion score,serum IgG,IgA,the number of IFN-γ secreted of lymphocytes.Our results shows that:As molecule adjuvants,all of FliC,CD40L,EDA have the effect to enhance the immune response and serum IgG titer of each groups can reach to 1:1600.The adjuvant effect of EDA is the best,the average of chicken weight gain in this group is(54.16±2.12)g,which has no significant difference(p>0.05)compared with the blank control group;and oocyst decrease ratio is 40.40%,more higher than other groups.The oocyst decrease ratio in double molecular adjuvant group(FliC-EmIMP1-CD40L)is 40.13%,which has no significant difference(p>0.05)compared with EDA group,but has significant differences(p<0.05)compared with single molecular adjuvant FliC and CD40L groups.As for the IFN-γsecretion,there were significant differences between three adjuvant group and the blank control group(p<0.05),and the results show that all the adjuvants can induce cell mediated immunity in chicken.The serum IgA level is very low in all groups,there were significant differences between three adjuvant group and the blank control group(p<0.05).The serum IgG level have significant differences(p<0.05)between the adjuvant groups and the control group,IgG level in EDA group is higher than other groups.Compared with FliC-EmIMP1 and EDA-EmIMP1,IgG level in FliC-EmIMP1-CD40L group have no significantly differences(p>0.05),and the EmIMP1-CD40L group have the least level of IgG.Conclusion:Molecular adjuvant,FliC,CD40L and EDA can promote the immune response of protein EmIMP1.All the adjuvant can induce the humoral and cellular immune responses in chicken,and then give the chicken some resistance against Eimeria maxima infection.Among them,The single molecular adjuvant vaccine EDA-EmIMP1 and the double molecular adjuvant vaccine FliC-EmIMP1-CD40L have the better protective effect.