Construction Of Live Vector Vaccine Of Recombinant Herpesvirus Of Turkey Expressing HA Protein Of H9N2 Subtype Avian Influenza Virus

H9N2 subtype avian influenza virus is a low pathogenicity,mild type of avian influenza virus,widely found in nature.The prevalence of H9N2 avian influenza virus in poultry production is still widespread in high-maternal antibodies.Infectious poultry mainly cause E.coli infection,accompanied by respiratory symptoms and bronchial obstruction and other diseases,which caused a huge economic loss to the chicken industry.At present,H9N2 AIV virus inactivated vaccine has a good immune effect,but such vaccines can not effectively induce cellular immunity and mucosal immune response,and it will be influenced by the maternal antibodies in the use of virus inactivated.HVT Fc-126 has been used as an MD vaccine since 1970.It has many advantages as a viral vector to express exogenous protective antigens:HVT is non-tumorigenic and safe to use;the hosts of HVT are narrow,only chickens and turkeys will be infected;chicken can produce viremia for a few weeks after vaccination,and can produce latent infection for life,the chicken can be stimulated to produce high levels of antibodies for the whole life;HVT can be made by freeze-dried technology,it's convenient for transport and storage;there is no need for adjuvant,the production cost is low.The development of recombinant vector vaccines that express the H9N2 AIV HA antigen with HVT can not only compensate for the deficiency of the inactivated vaccine,but also cause latent infection and cause lifelong immunity.In this study,a recombinant rHVT-HA vaccine can provide technical support for effective prevention and control of H9N2 AIV and MD.In this study,we constructed HVT bacterial artificial chromosome,using the bacterial artificial chromosome(B AC)as carrier,using xanthine-guanine phosphoribosyl transferase(Eco-gpt)as the selection marker gene,and then co-transfecetd plasmid and HVT infected cells into primary chicken embryo fibroblasts(CEF),after 5 rounds of selection in medium containing mycophenolic ac id,xanthine and hypo xanthine,we obtained purified recombinant virus.The correct clone was identified by PCR and restriction enzyme digestion,and then was transfected into CEF to rescue HVT recombinant virus.Using the RED/ET recombination technique and ccdB reverse counter-selection technique,we are trying to develop a biologically live vaccine based on the HVT BACs.firstly,the DH10B competent cells carrying the HVT BACs were transformed with expression plasmid pREDET,theninduced by the addition of L-arabinose and L-rahmnose.The linear ccdB-amp counter-selection cassette flanked by 50bp long homology arms was electroporated into the competent cells.Secondly,in the same way the ccdB-amp cassette was replaced by the non-selectble target DNA-hemagglutinin gene.Finally,we obtained many B ACcolonies of which the HA genes of the AIV was inserted into the US21ocus to be modified of HVT,then,the rescued virus was designated as rHVT-HA.