Construction And Immunogenicity Analysis Of Recombinant Baculovirus Expressing Major Antigens Of Duck Tembusu Virus

In 2010,Duck tembusu virus(DTMUV)emerged in China and caused extensive epidemics in egg-laying and breeder ducks.The affected ducks showed a severe drop in egg production and paralysis.Such virus can impact other po ? Ltry or birds as well,such as chicken,geese and sparrow.Rate of illness was usually high(up to 100%).egg production dropped from 95%to 5%.secondary infection mortality rate reach to 30%.In 2010,duck tembusu virus disease cause enormous economic losses in China.Some studies find TMUV as a possible infection in duck industry workers,and confirmed the emergence of TMUV in humans.Structural proteins of duck tembusu virus include C protein,M protein and E protein.Both protein M and E are membrane protein are membrane proteins.Studies have shown that M protein has significant importance for E protein structural stability and it can promote the induction of protective immunity.Most studies of flavivirus showed that E protein has a great conservatism and immunogenicity.Such protein can be used as the preferred gene for subunit vaccine and genetic engineering vaccine construction.Below is more detailed imformation on this study:1?Preparetion of polyclonal antibody prM and EPrM and E protein were expressed in the E.coli After purification,prM and E proteins were used to immune the rabbits according to the procedure in the methods.Polyclonal antibody with high titer were harvested from the serum.2?Construction of recombinant Baculovirus expressing prM-E proteinPrM-E gene was cloned into DNA of Baculovirus(rBac-prM-E)using the Bac-to-Bac system.After transfection of sf9 cell with recombinant bacmid rBac-prM-E,sf9 cell get lesions and recombinant Baculovirus were harvested.The protein expression were confirmed by Western blot,immunofluorescence,and the form ? Lation of duck tembusu virus like particles(VLPs)were confirmed by electron microscope.3?Purification of prM-E VLPs and the preparation of subunit vaccine for animal testPrM-E VLPs from sf9 cell and c ? Lture were purified by sucrose gradient centrif ?gation,It is mixed with adjuvant and used to immune cherry valley duck.The res ? Lts showed that the vaccine can induce specific antibodies against E antigen.4?Preparation of subunits vaccine using the prM-E expressing sf9 cell clasmatosis productsCrushing the sf9 cell which can expressing prM-E proteincentrif ? ging it and harvest the supernatant.It is mixed with adjuvant and used to immune laying ducks.Twenty-nine days latter,the laying ducks were challenged with duck tembusu virus DF2.The res ? Lts showed that shrivel ovary were observed in the positive control.There were two or three duck in the testing group one or testing group two,Therefore,the protection rate of the subunit vaccine in this study is 40%or 60%.5?Construction and detection of recombinant Baculovirus expressing E?proteinIn this study,a recombinant Baculovirus expressing E? protein were constructed and the western blot showed that E? protein can expressed both in the sf9 cell or in the c ?Lture in the form of soluble protein.