Expression And Purification Of Infectious Bovine Rhinotracheitis Virus GD Gene In CHO-K1 Cells

Infectious bovine rhinotracheities(IBR)is a respiratory disease caused by infection bovine rhinotracheities virus(IBRV).After IBRV infects cattle,the main clinical symptoms are dyspnea,accompanied by fever,loss of appetite,mastitis,conjunctivitis,miscarriage,and inflammation of the reproductive tract.Although the fatality rate of IBR is not high,the acute infection of IBR in cattle will cause immunosuppression and often secondary infection with other pathogens,thereby aggravating the disease and increasing the mortality rate.As the envelope glycoprotein of IBRV,gD protein plays an important role in the process of IRV invasion of cells.It can also induce the body to produce strong humoral and cellular immunity,produce protective antibodies,and the monoclonal antibodies prepared against gD have neutralizing activity,Can neutralize the virus.Therefore,gD protein can be used as a candidate target protein for developing diagnostic reagents,preparing subunit vaccines and antiviral drugs,and has important research significance for the prevention and treatment of IBR.In this study,to obtain the gD protein,according to the IBRV gD(accession number:NC＿001847.1)gene sequence published by Gen Bank,the TMHMM Server-2.0 online software was used to predict and find the target gene without a transmembrane region,and the Signal P-4.1 online software was used to predict and find the target gene With signal peptide,according to the polyclonal restriction site of pSecTag2 A eukaryotic expression vector,appropriate restriction sites are added on both sides of the target fragment,and gene synthesis is performed after the codon preference optimization of the CHO expression system.The target gene was digested with the eukaryotic expression vector,and the pSecTag2A-gD recombinant eukaryotic plasmid was successfully constructed.The pSecTag2A-gD was transfected into CHO-K1 cells,and the cell supernatant was collected for seven consecutive days.First,Western Blot was used to detect the His tag,and the highest expression level was determined 48 h after transfection,and then the target protein was detected by gD monoclonal antibody.Reactogenicity,and finally the gD protein was purified by nickel column affinity chromatography.The results are as follows:1.The results of agarose gel electrophoresis after double enzyme digestion showed that the pSecTag2A-gD recombinant eukaryotic plasmid has been successfully constructed.2.After transfection into CHO-K1 cells,collect the cell supernatant,use anti-His monoclonal antibody as the primary antibody,and use Western Blot to successfully detect His tag;use gD monoclonal antibody as the primary antibody,Western Blot results show that the target protein is The ability of gD monoclonal antibody to bind indicates that gD protein has reactogenicity.3.Purify the gD protein by nickel column affinity chromatography,and successfully purify the target protein.The gD protein expressed in this study lays the foundation for the establishment of later detection methods for IBRV and the development of vaccines.