Development And Characterization Of Single Chain Antibody Against GD Glycoprotein Of Bovine Infectious Rhinotracheitis Virus

Infectious bovine rhinotracheitis virus,also known as Bovine hepersvirus I(BHV-1),is the pathogen of bovine infectious rhinotracheitis(IBR),Since the 1950 s,it was reported in the United States at first,Nowadays it was widely distributed all over the world,it is one of the most important causes of economic loss in the cattle industry.gD is one of the major glycoproteins of the bovine infectious rhinotracheitis virus and it can induce neutralizing antibodies.IBRV established Latent infection in the rigeminal ganglion would result in persistent infection and virus shedding,which makes it difficulty to eradicate completely.it is difficult to antibody to eliminate the virus in the cells,with the development of single-chain antibody this problem would be resolved easily,single-chain antibody is one of small sized recombinant antibodies,it was composed of the heavy chain variable region and the light chain variable region of the antibody through a Linker peptide.It can bind the virus in the cells.VH gene and VL gene were amplified from the cDNA of lymphocyte hybridoma,then VH gene and VL gene were integrated with a Linker by SOE-PCR.ScFv was expressed in E.Coli and Sf9 cell.the ScFv gene was cloned into pET28 a plasmid to construct an expression vector,then transformed into Escherichia coli BL(DE3).The expressed single chain antibody was 30 kD,and it can specifically bind to anti-His antibody and it was expressed as inclusion body.the single-chain antibody fusion protein was obtained after degeneration,purification and renaturation of the expressed product.Western blot,antigen capture ELISA,IFA show that the single chain antibody can bind to IBRV specifically.The virus neutralization test showed that the single chain antibody can react with the IBRV and can inhibit the replication of the virus in bovine kidney cells(Madin-darby Bovine Kidney,MDBK)effectively.When the ScFv was expressed by Sf9 cell,the gene was cloned into the baculovirus vector pFastBac-Lic-Bse,which was transformed into Escherichia coli DH10 Bac competent cells to construct a recombinant bacmid rBacmid-VH-VL.Finally,ScFv was expressed by transfected rBacmid-VH-VL DNA into insect Sf9 cells,its molecular weight is about 30 kD.The single-chain antibody can bind to anti-His antibody and gD protein specifically.Indirect immunofluorescence assay showed that the single chain antibody could recognize IBRV infected with MDBK,but its affinity was not strong as ScFv that expressed by E.Coli.The single chain antibody was expressed in E.Coli and Sf9 cell.when the ScFv was expressed by Escherichia coli,the affinity of bind to bovine infectious rhinotracheitis virus and gD protein was stronger.because of its simple preparation and low cost,the single chain antibody will provide a basis for development of diagnostic methods for bovine infectious rhinotracheitis and effective therapeutic agents.