Construction Of A Recombinant Dev Expressing The Vp1Gene Of Dhv-1and Preliminary Study Of The Recombinant Virus Bionomics

Duck viral hepatitis is an acute and highly contagious disease of young ducklings characterized by high mortality and liver lesions with haemorrhagic spots. VP1protein is the major Virulence determinants which contains multiple enpitope of blinding cells and can induce the immune response and neutralizing antibodies. Duck viral enteritis (DVE), also known as duck plague, is an acute, contagious and lethal disease of ducks, geese and swans, caused by duck enteritis virus (DEV). In this study, DEV was used as a recombinant viral vector, and we constructed a recombinant virus of DEV which can express VP1protein of DHV and lay the material foundation for the duck hepatitis virus genetically engineered vaccine.VP1gene was amplied by RT-PCR and cloned into pcDNA3.1(+),then we amplied VP1expression cassette including pCMV and BHG ploA by PCR. EGFP expression cassette, GPT expression cassette and homologous arms gene were amplied by PCR. Then EGFP expression cassette, VP1expression cassette, the homology arms gene and GPT expression cassette were cloned into pMD18-T vect in turn by restriction sites and got recombinant virus transfer vector pMD18-T-VP1-GPT-EGFP. pMD18-T-VP1-GPT-EGFP and DEV DNA were cotransfected with the second generation duck embryo fibroblasts. A pure virus was obtained after homologous recombination and pressure screening. The purified virus were identified with PCR, RT-PCR,Western blot and IFA and named as DEV/ΔgG/VP1. DEV/ΔgG/VP1and DEV has the same structure according to electron microscopy.Then DEV/ΔgG/VP1particles were reacted with rabbit anti-DHV VP1polyclonal antibody, the results of immune electron microscopy showed that VP1protein could be expressed in the virus particle surface.100TCID50DEV and DEV/ΔgG/VP1were inoculated in DEF respectively, and determined the title of virus collected by the different time, the result showed that the time of DEV/ΔgG/VP1titer reached the peak of was96h, which was later than DEV. The lack of gG protein has no effect on viral replication,but its proliferation time lagged relativly. The plague diameters of DEV and the recombinant virus did not have significant differences,which showed that the insertion of foreign genes did not affect the spread of the virus in cell. DEV/ΔgG/VPl was passaged for20rounds, then identified VP1gene by PCR. VPl gene can be amplified, VP1protein of the20th generation of recombinant virus could be detected by Indirect immunofluorescence assay. The rescult showed that VP1gene can exist in the recombinant virus stablely.