Preparation And Identification Of Bovine Infectious Rhinobronchitis Virus GD Protein Specific Single Domain Antibody

Infections bovine rhinotracheitis(IBR)is caused by Infections bovine rhinotracheitis virus(IBRV).Under normal circumstances,IBRV is prone to repeated infections,which makes the disease difficult to remove and seriously harms the cattle industry.At present,vaccine immunization is the main measure to prevent and treat IBR,but inactivated vaccine can not cause long-term continuous humoral immunity,and the attenuated vaccine has the risk of residual virulence and re-strength.In order to effectively prevent and control IBR,it is necessary to develop effective diagnostic antibodies.In this study,IBRV gD gene was cloned,ligated into prokaryotic expression vector,transformed into E.coli,induced by IPTG,purified and identified to obtain IBRV-gD protein;then IBRV was used as antigen to immunize adult healthy Bactrian camel,whole blood was collected and extracted.The total RNA of lymphocytes was reverse transcribed,and the VHH gene fragment was amplified and ligated into the vector pCANTAB5E,which was transformed into E.coli TG1 competent state,and the IBRV VHH antibody library was successfully constructed.The IBRV antibody library capacity was identified as 7×107.After colony identification,the IBRV antibody library conversion efficiency was 75%.The gD protein was used as a screening antigen,and the phage display technology was used to screen from the IBRV VHH antibody library,and a positive clone with strong relative binding ability was selected to construct an expression vector,which was induced to express and purify,thereby obtaining high binding activity of IBRV gD.Specific single domain antibody.The single domain antibody(IB68)can be identified by ELISA to produce antigen-antibody binding reaction with both IBRV or gD protein,and has high antigen binding activity.Western-Blot assay confirmed the immunological reactivity of the single domain antibody(IB68)prepared in this assay.It lays the foundation for the development of rapid diagnosis reagents for IBRV in the future.