Expression And Immunogenicity Of The Duck Tembusu Virus E Protein In Insect Cells

Duck tembusu virus disease is an emerging infectious diseases which can lead to a severe drop in egg production of the laying ducks and growth retardation.Meat-type duck and laying duck can be infected by the virus.This disease has caused a serious economic loss to the poultry industry in China.Currently,the prevention of DTMUV mainly rely on the inactivated vaccine and attenuated vaccine immunization.It has been shown that DTMUV E protein is the most important immune antigen.Which can induce protective neutralizing antibodies.In this thesis,DTMUV E protein was expressed in Sf9 cells,and the immunogenicity the recombinant E protein was also examined.Firstly,DTMUV E gene was cloned from pMD19-E and inserted into pFastbac,then the recombinant plasmid pFastbac-E was transfected into DH10 Bac compent cells.After amplification,the purified pFastbac-E wastransfected into Sf9 cells with Cellfection.At last,the recombinant viruses(rvBac-E)were covered.After passaged four generation in sf9 cells,the titre of rvBac-E was TCID50:10-5.5/0.1mL.The results of Western blot and IFA showed that DUMTV E gene were well expressed in sf9 cells,and the expressed recombinant E protein could reacted with the specfic antibody against DTMUV.The animal experiment was conducted for examing the immunity of the recombinant E protein.The 7-day-old cherry valley ducks were divided into 4 groups(G1-G4): the duckings of group1(G1)was immunized with the recombinant E protein,named experimental group,the duckings of group 2(G2)was immuned with wtBac,which was as negative Bacmid group;the duckings of group 3(G3)was immunized with ther killed vaccine(AH-F10 strain),which was as the positive control group;The last group was immunized with PBS as the negative control group.The specific antibodie against E protein s,serum neutralized antibody,IL-4 and IFN-? cytokines were detected in every week after immunized.The lymphocyte proliferation test was detected after immunized 4 weeks.The results showed that the levels of the specific antibodies against E protein,serum neutralizing antibody,IL-4 and IFN-? were significantly increased in G1 ducklings after immunized 2 weeks,compared with the negative control and Bacmid group;lymphocyte proliferation test showed that the lymphocyte SI value in G1 ducklings was significant difference with negative group and Bacmid group.After immunized 4 weeks,all the cherry valley ducklings were challenged with DTMUV AH-TL15 strain(ELD50 = 10-2.38 / 0.1 mL).The results showed that the protection of ducklings in the experimental group was 80%.The pathological changes of ducklings in the experimental group and the positive control group could not be observed.On the contrary,ducklings of the negative control group and the Bacmid group showed obvious pathological changes in the liver,spleen,pancreas and the brain,which were the typical pathology of DTMUV.In summary,we successfully expressed DTMUV E protein in sf9 cells.And the recombinant E protein could effectively induce humoral immunity and cellular immunity in cherry valley ducklings,which could protect cherry valley ducklings against virulent DTMUV attack.These results suugged that the recombinant E protein expressed in sf9 cells has the potential as a new vaccine against DTMUV infection.