Expression Of Novel Duck Reovirus σC Gene In Insect Cells And The Immunogenicity Preliminary Study Of Expressed Protein

In recent years, waterfowl reovirus disease has been increased,seriously affected the development of water industry.Since 1997 a white-spots of livers diseases which with soft feet, diarrhea, necrosis of liver white as the main characteristics was appeared in muscovy duck popular culture zones, since 2005 many varieties ducks occured the necrotizing hepatitis which, the main characteristics is there are hemorrhage and necrosis appeared in the heart, spleen, liver, bursa and other organ, researches shown that the pathogeny caused these diseases is avian reovirus. (ARV).Study shown that ARV have changed in epidemiology, clinical symptoms and pathological changes, clinical characteristics, biological characteristics, genome characteristics and sequence homology etc. A novel duck-pathogenic orthoreovirus subsequently was isolated from affected ducks in our lab.ARV cC protein is able to attach to avian cell and can stimulate the body to produce specific neutralizing antibody, the attachment of aC to cultured cells can be competed by purified reovirions and the attachment of avian reovirions to permissive cells can be blocked by preincubating the cells with protein,So the σC protein can induce effective immunoprotection.up to now there is no biological products for the prevention and treatment of the disease, the next step is to find a way how to diagnose the disease quickly and efficiently and develope effective vaccine.This study prepare the polyclonal antibody from the rabbit immunized with purified recombinant prokaryotic expression aC protein, constructed a recombinant baculovirus BV-σC, prepare ofgenetically engineering vaccine, investigated the vaccine immunogenicity is, conduct the challenge assay and observe the immune protection. The main research content is as follows:1. Prokaryotic expression and polyclonal antibody preparation of aC protein of novel duck reovimsσC gene was cloned into pET-30a(+) and expressed in E.coli BL21(DE3). SDS-PAGE and Western blot analysis showed that the expressed product was about 34Ku and had good immunological activity.The polyclonal antibody was obtained from the rabbit immunized with purified recombinant protein and its titer was about 1:20000 by detection of indirect ELISA. Indirect immunofluorescence assay also demonstrated that the polyclonal antibody reacted specially with the native NDRV σC protein.2.Expression and identification of NDRV σC gene in sf9 cellsInsert NDRV oC gene into baculovirus vector pFastBacTM1, through homologous recombination, blue white spot selection, obtain the recombination Bacmid -σC and transfect sf9 cells, and transfect empty Bacmid as control.Performing a Viral Plaque Assay to determine the titer of baculoviral stock, the titer of this viral stock is 2 x107pfu/ml. By SDS-PAGE proved the aC has successfully expressed;Western blot, IFA results show that the aC protein possesses immunogenicity.3. The immunogenicity preliminary study of NDRV σCSf9 cells were infected by recombination baculovirus and product a amount of σC proteinin.Preparing vaccine with the protein and immune 1 week duckings, two weeks after the first immune,immune again with larger doses.Results show that the experimental group can induce neutralizing antibodies after 2 weeks, after strengthening immune antibody levels continue to rise and can achieve 1:32 antibody neutralizing titer. The experimental results show that the recombinant baculovirus vaccines induce effect humoral immunity.Two weeks after the second immune, all ducks from the three groups were challenged with 0.5ml NDRV.The result showed that immune group obtain good protection, protection rate could be achieved at 70%.The reslt of challenging indicated that the vaccine induce an immune reponse and resisted the NDRV at a lethal dosage,providing a foundation for the development of safe and effective vaccine against NDRV.