Construction Of Recombinant Adenovirus Of VP2 Protein Of Infectious Bursal Disease Virus JS Strain And Biological Characterization Of The Recombinant Virus

Infectious bursal disease virus (IBDV) is the causative agent of a contagious disease in chickens of infectious bursal disease (IBD). The virus infection in young chickens at 3-12 weeks age can increases susceptibility to other diseases. VP2 protein of IBDV is a major structural protein, which plays an important role in virus assembly and can induce neutralization antibody against IBDV. So the VP2 gene is significance for the vaccine development of IBDV. .A pair of specific primers for the VP2 gene of IBDV was designed according to the gene sequence of a vvIBDV (Accession No.M64285) strain. The VP2 gene was amplified by the reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis showed that the amino sequence of VP2 gene of JS strain is high homology with the vvIBDV strains (94.8-99.1%). The VP2 gene was subcloned into pcDNA3.1/zeo(-) vector and then transfected to HEK-293 cells . The expression protein of VP2 in HEK-293 cells was identified by immunofluorescent assay (IFA).The result demonstrated that the VP2 gene can expressed in eukaryotic cells.In order to develop recombinant adenovirus vaccine, the VP2 gene was inserted into the pShuttle-CMV plasmid to obtain the pShuttle-CMV-VP2. The linearized pShuttle-CMV-VP2 plasmid was transformed into E.coli BJ5183 which containing backbone vector pAdEasy-1 to make the homologous recombination happen. The recombinant plasmids were linearized and then transfected into HEK-293 cells with transfection reagent Lipofectamine 2000 to get the recombinant adenovirus. After passed 3 generations in HEK-293 cells, the recombinant virus was purified by plaque purification. The titer of recombinant virus was 107.8 TCID50/mL. The expressing of VP2 protein in HEK-293 cells infected with the recombinant virus rAd-vp2 can be detected by indirect immunofluorescent assay (IFA) and Western-blot with monoclonal antibody to IBDV VP2. Balb/c mice immunized with HEK-293 cells infected with rAd-vp2 could elicit neutralizing antibodies to IBDV. These results suggested rAd-VP2 might be an attractive candidate vaccine for preventing IBDV infection in future.