Constrution And Identification Of Recombinant Turkey Herpesvirus Expressing HA Of Avian Influenza Virus H5N1

H5N1 avian influenza(H5N1 AI)has become a serious threat to the poultry industry,and received extensive concerns worldwide.Inactivated vaccine has been the primary method of prevention and control of the disease,but can provide poor protection against the pathogen.Furthermore,there might be a risk for live virus transmission during the preparation of the vaccine.The development of genetically engineered live vector vaccine has recently become a research hot spot avian influenza due to its unique advantages in the prevention of infectious diseases.HA gene is therefore the preferred antigen gene in the development of genetically engineered vaccine against avian influenza.Commercialization of turkey herpes virus(HVT)vector has become a good vector for live vaccines against poultry viruses HVT vector has higher immune efficacy compared with conventional vaccines..The herpesvirus bacterial artificial chromosome(BAC)has become a powerful method for the development and improvement of recombinant herpesvirus vaccines.In this study,a recombinant HVT expressing HA of a highly pathogenic AIV(H5N1),named HVTBAC-H5 was constructed through the insertion of HA expression cassette into a non-essential region of HVT genome.The current study has provided a basis for future development of genetically engineered live vector vaccine against AIV.In this study,an HA gene was designed and synthesized based on the sequence data of H5N1 2.3.2.1 published in Pubmed,and an HA expression cassette was constructed containing the HA gene,CMV promoter and poly A terminator.The HA expression cassette was amplified by PCR using a pair of primers containing the homology arms bordering the insertion sites in the HVT genome,and inserted into the non-coding region between UL54 and UL55 on the BACHVT-G by the EN PASSANT method.The obtained recombinant BAC(BAC HVT-HA KAN+)was confirmed by PCR and sequencing,and subjected to a second recombination to knock-out the Kan resistance gene.The obtained virus vector,named BACHVT-G-H5,was confirmed by PCR and RFLP.The BACHVT-G-H5 DNA was extracted,with PCR fragments containing the homology arms and transfected into chicken embryo fibroblasts(CEF)by the calcium phosphate-based method in order to rescue the viros.The recombinant virus HVTBAC-H5 containing the BAC sequence was successfully rescued as indicated by the green fluorescent plaques under UV light.The genomic DNA of HTVTBAC-H5was extracted,and HA expression cassette was amplified and sequenced.It was shown that the designed HA expression cassette had been successfully inserted.Hemagglutinin test and western blot showed that the HA was properly expressed and specifically bound to the H5N1 positive serum.In summary,this study has successfully constructed a HA recombinant HVT.The current method has developed an efficient method for the construction of HVT recombinant virus based on BACHVT-G and EN PASSANT,which provides a basis for the development of novel HVT-vectored vaccine against AIV.