Construction And Immunological Study Of Adenoviral Vector Vaccine Containing Duck Tembusu Virus E Gene And Duck IL-2 Gene

Since the first DTMUV disease infecting China ducks was reported in April 2010,it has rapidly spread in China's coastal areas and caused huge economic losses.In this study,in order to lay the foundation for the development of recombinant adenovirus vaccines for duck tembusu virus disease,we constructed a recombinant adenovirus expressing fusion gene of duck tembusu virus E and duck interleukin 2.Results were as follows:1.Construction and identification of the recombinant adenovirus rADV-E-IL-2 and rADV-E.A 1733 bp fusion fragment that ligating the 1353bp E gene of duck tembusu virus and the 350 bp duck IL-2 gene were cloned to the recombinant adenovirus shuttle plasmid to construct pshuttle-E-IL-2,simultaneous construction of the viral E gene plasmid pshuttle-E as a control.Both of them were co-transfected into the HEK293 cells with the backbone vector respectively,and the recombinant adenoviruses rADV-E-IL-2 and rADV-E were identified by PCR and western blot.The virus can be serially passaged more than 20 generations in HEK293 cells,and have genetic stability.After purification,the titer of rADV-E-IL-2 reached 4.0×10¹⁰PFU/mL,and the titer of rADV-E reached 6.0×10¹⁰PFU/mL.2.Protective experiment of recombinant adenovirus on ducks.80 1-day-old ducklings were randomly divided into five groups?rADV-E-IL-2 group,rADV-E group,rADV-N group,WF100 group,and PBS group?and subcutaneously infected recombinant adenovirus rADV-E,recombinant adenovirus rADV-E-IL-2,empty recombinant adenovirus rADV-N,commercial duck blubber vaccine WF100 strain and PBS at 7 days and 21 days of age.The results showed that specific IgG against DTMUV were induced in the rADV-E-IL-2 group and the rADV-E group,rADV-E-IL-2 group reached at the highest peak?1.39?at 35 days after immunization which had significant difference compared with other immunized groups?P<0.05?.Both of the rADV-E-IL-2 group and the rADV-E group had neutralizing ability,and their neutralizing antibody titers reached the highest level at35 days after immunization.The potencies of the rADV-E-IL-2 group and rADV-E group were 1:55 and 1:44 respectively.Both the rADV-E-IL-2 group and the rADV-E group significantly increased the proliferation of peripheral lymphocytes which the proliferation index were 3.13 and 2.69 respectively.Compared with rADV-N group and PBS group,the difference was extremely significant?P<0.05?.Compared with the rADV-C group and the PBS group,serum IL-2 levels?94.78 pg/mL,76.18 pg/mL?,IL-6 levels?66.43 pg/mL,59.45 pg/mL?,and IFN-?levels?201.51 pg/mL,128.61 pg/mL?of rADV-E-IL-2 and rADV-E groups had significant difference?P<0.05?.In the challenge protection experiment,ducklings immunized with the rADV-E group and the rADV-E-IL-2 group had no clinical symptoms,and the spleens were free of lesions.In the rADV-E-IL-2 group,the copy number of DTMUV in the spleens was 10^1.22copies/?L,and the copy number was10^1.81copies/?L in the rADV-E group.Both rADV-E group and rADV-E-IL-2 group were significantly lower than those in the rADV-N and PBS groups?P<0.05?.