Preparation Of Anti-BoHV-1 Monoclonal Antibody And Identification Of GD Protein Epitope

Bovine Infectious Rhinotracheitis Virus(IBRV)is also known as Bo HV-1,aninfectious bovine rhinotracheitis(Bovine Infectious Rhinotracheitis,IBR)pathogen,which is one of the most important viral pathogens of bovine respiratory diseases.It could cause not only respiratory tract inflammation,but also conjunctivitis,abortions and neurological symptoms.In addition,Bo HV-1 is characterized by periodic activation of latent infection of trigeminal ganglion,which leads to greater difficulty in clearing bovine infectious rhinotracheitis(IBR)and affects the development of breeding industry.At present,the traditional commercial vaccine in China has potential risks,so the development of new vaccine and antibody diagnostic preparations has positive significance for the prevention and treatment of IBR.In this study,hypercentrifuge concentration of Bo HV-1 Barthanu /67 strains was used for immunogen in BALB/c mice at the age of 6-8 weeks.Two strains of monoclonal antibody which were named as 3C1 and 4F3 respectively were screened by indirect immunofluorescence method and ascites were prepared.The heavy chain of 2 monoclonal antibody strains was Ig Gl and Ig A,and the light chain was kappa.The titers of superscript and ascites were determined by indirect immunofluorescence method.The titer of monoclonal antibody 3C1 was 1:40 and 1:12 800,and the titer of monoclonal antibody 4F3 was 1:40 and 1:1 600,respectively.Indirect immunofluorescence and Westernblot verified that 3C1 could bind to g D protein in a specific way without cross-reacts with BRV,BVDB and PRV,which showed good specificity.The neutralization activity of 3C1 antibody in MDBK cells was tested for bohv-1 infection,and the sum titer was calculated as 1:1409.The linear epitope of g D protein was identified by the preparation of g D monoclonal antibody.The truncated g D recombinant protein was constructed,and finally connected to the expression vector after annealing of the upstream and downstream primer for expression,and the epitopes identified by 3C1 were defined by Westernblot test.It showed that 3C1 recognizes APRVTVYVD(aa23-31)of g D protein.VTVYVD(aa23-26)fragment was identified as the minimum amino acid sequence by truncating from N and C terminus respectively.Epitopes identified in this study were highly conserved in the 15 classical herpes viruses.In summary,this study successfully prepared two antibodies,one of which was anti-Bo HV-1g D protein MAb with neutralizing activity,and identified the epitope of g D protein.g D protein of Bo HV-1 neutralizes in cells and produces a protective immune response to Bo HV-1 infected cells.Therefore,Therefore,the preparation of neutralizing active antibodies and the positioning of epitopes on g D will contribute to enhance our comprehension of the protein structure to elucidate the pathogenesis of Bo HV-1,and provide an important basis for the development of alternative vaccines and antibody treatment and diagnosis techniques for various diseases related to this virus.