Construction And Preliminary Immunogenicity Evaluation Of Recombinant HVT Expressing Newcastle Disease Virus F Protein

The recombinant vector vaccine is a live vaccine that uses genetic engineering technology to construct a virus or bacteria into a vector,and then inserts the foreign gene into the vector and expresses it.Such vaccines can induce the body to produce a wide range of immune responses,and recombinant live vector vaccine can simultaneously express multiple antigens and have the potential to become multivalent or multi-linked vaccine,which is one of the main directions of current and future vaccine development.Marek's disease virus(MDV),as a member of the herpesvirus family,has a large genome and can be inserted into foreign genes.The ideal viral vector for recombinant vaccines.Serum type 3 MDV is Turkey herpesvirus(HVT),which has been used as a vaccine to prevent Marek's disease for more than 50 years,and HVT is also often used as a vector to express foreign genes.HVT as a vector has many advantages: HVT can be vaccinated with 1-day-old chicks without interference from maternal antibodies;HVT is not zoonotic and safe to use;HVT can continue to stimulate the production of antibodies after one vaccination without multiple booster immunizations;compared to other whole virus vaccines,HVT only expresses protective antigen component does not have the risk of returning to virulent strain,and it is convenient for immune monitoring.Newcastle disease(ND)is a dangerous infectious disease of poultry caused by a virulent strain of Newcastle disease virus(NDV),which has a very high mortality rate,caused inestimable losses to the poultry industry.In recent years,the incidence of atypical Newcastle disease has continued to increase,showing varying degrees of endemicity,and ND has also occurred in immunized flocks.The main reasons for this phenomenon are the high level of maternal antibodies and the genotype mismatch between the ND epidemic strain and the vaccine strain.Therefore,the development of new and effective vaccines is great significance for the prevention and control of ND.This study aims to establish a HVT fosmid rescue system that can efficiently rescue virus,and then insert the NDV epidemic strain F gene expression cassette of the into it,construct a recombinant viral disease to evaluate its immune efficacy.This study have important application value,and provide technology supports for the effective prevention and control of Newcastle disease and Marek's disease.In this study,the HVT FC126 strain gene segments were inserted into the Fosmid vector pCC1 Fos to construct the FC126 strain genomic fosmid library;on this basis,the fosmid combinations were selected and co-transfected with chicken embryo fibroblasts to establish the HVT FC126 vaccine strain multi-segment fosmid rescue system.Through this rescue system,the NDV gene type VII strain LHLJ/01/06 strain F gene expression cassette was inserted between the HVT065 and HVT066 genes in the HVT genome to construct the recombinant virus rHVT-NDV-F.PCR and sequencing identification showed that the F gene expression cassette was successfully inserted into the FC126 strain genome.Indirect immunofluorescence assay show that the recombinant virus rHVT-NDV-F can express F protein.There is no significant difference in the replication ability of rHVT-NDV-F recombinant virus and its parental virus HVT FC126 strain in vitro.After continuous passage,the F gene expression cassette inserted in the recombinant virus rHVT-NDV-F genome can stably exist and express F protein.The immune protection test of rHVT-NDV-F recombinant virus found that the protection rate of the recombinant virus rHVT-NDV-F against the NDV gene type IX virulent F48E9 strain is 80%,and that of the NDV gene type VII epidemic strain LHLJ/01/06 the protection rate is 70%.In summary,this study constructed the HVT FC126 strain virus rescue system,and the recombinant virus rHVT-NDV-F is capable of expressing the NDV gene type VII strain F protein;the in vitro replication ability of rHVT-NDV-F has no significant effect on the parent And it has satble genetic stability;the vaccine strain can provide better protection to the currently epidemic NDV VII strain,and has the potential to be used as a live ND recombinant HVT vector vaccine.