| Duck virus hepatitis (DVH) caused by duck hepatitis virus typeâ… (DHV-â… ) is a highly mortal infection disease. DHV-â… is prevalent in China, which lead large economic loss for duckery. To date, no much research of protein function for DHV-â… has been reported, only the genomic structure and proteidic organization predicted, which greatly hindered the relevant research. In this study, structural protein VP1,VP0 and VP3 gene of DHV-â… QV strain was cloned and were expressed in eukaryotic expression system, which is important for the research of molecular epidemiology of DHV-â… and new type vaccines for DHV-â… .In this study, three pairs of specific primers were designed and synthesized according to gene sequences of DHV-â… strains available in GenBank. Genome sequence of DHV-â… QV strain was amplified by reverse transcription polymerase chain reaction (RT-PCR). The products of RT-PCR were cloned into the pFastBacHTB vector, and then transformed to competent E.coli Top10 cells. Recombinant plasmids were dectected by digestion with endonuclease enzyme and sequencing. and transformed into competent cells of DH10Bac and the position colonies were screened by blue/white spot selection and verified by PCR. Recombinant bacmid were transfected into sf9 cells using Cellfection reagent to obtain the product of recombinant viral stock.The biological activities of recombinant protein were analyzed by SDS-PAGE and Western-blot.Results showed that structural protein VP1,VP0 and VP3 gene of DHV-â… QV strain was cloned. rBacmid-VP1, rBacmid-VP0 and rBacmid-VP3 were triumphantly established. The results of SDS-PAGE and Western-blot demonstrated that recombinant protein VP1,VP0 and VP3 were expressed in eukaryotic expression system and exhibited specific immunological activity with DHV-â… masccline blood serum, which suggested that these protein have good biological activity, respectively 36KD, 38KD, 30KD. These results can good for development of genetic engineering vaccine with expression protein. |
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