Development Of Monoclonal Antibodies Against BHV-1gB And Establishment Of Double-antibody Sandwich ELISA Method

Infectious bovine rhinotracheitis (IBR) is an acute, pyretic and contagious disease, caused by bovine herpesvirus-1(BHV-1). It’s a B listed disease defined by office international epiz-ooties (OIE) and a key check item in the import animals and the international animal trade in our country. The main infection target is cattle, especially the beef cattle, secondly cows. Once infected with BHV-1, the cattle will be a carrier for lifetime. ELISA is a sensitive and rapid method to detect BHV-1, which can detect BHV-1in recessive patients in time, prevent the spread of virus in the herd and lower the economic loss.BHV-1is a member of varicella genera in α herpes subfamily. The BHV-1genome is double strand, linear DNA. The length of nucleotide sequence of genome is about137Kb, contains about70encoding genes, with a layer envelope containing lipid in the most superficial. The gB gene is about2940bp, and highly conservative, which is necessary for the virus infection and is one of the main antigen protein to stimulate the host immune response. This study engaged in the prokaryotic expression of gB envelope protein in E. coli and prepared the monoclonal antibody against BHV-1.Part one:Prokaryotic expression of gB gene of BHV-1in E. coli. According to the sequence of BHV-1Colorado1strain in GenBank. A pair of primer was designed to amplify the gB gene by PCR which yielded a specific band of585bp. pET-22b(+) was chosen as prokaryotic expression vector with histidine tag to construct the recombinant protein. After transformation of recombinant plasmid pET-22b-gB into E. coli BL21(DE3), a fusion protein about26KDa is obtained which contained a tag protein of5.2KDa. The supersonic lysate was denatured and renatured by urea and purified by Protino Ni-TED2000packed columns kit. The result of Western-blot showed that the fusion protein had good immunogenicity with BHV-1gB positive serum.Part two:Development and primary application of monoclonal antibodies against BHV-1gB. After the proliferation in MDBK cell, BHV-1is concentrated by polyethylene glycol6000and purified by20%sucrose cusion centrifugation. Finally, the virus suspension got by dialysis was used to immunize6weeks old Balb/C mice. Fusing SP2/0myeloma cells and spleen lymphocyte of immuned Balb/C mice, three monoclonal antibodies against gB were detected by indirect ELISA (use the purified virus and pET-22b-gB protein for screening antigen). Three hybridomas cell lines named2G4,2G6,3E9were obtained with the titers of128000,128000,64000. The results of Western-blotting and IIF showed that the monoclonal antibodies can react with purfied BHV-1virus typically and had no cross reaction with bovine rotavirus (BRV), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza3virus (BPIV-3). The subclass of2G4,2G6,3E9were all determined as IgM by method of capture ELISA. Because of high specificity of monoclonal antibodies and sensitivity of double-antibody sandwich ELISA method, improved double-antibody sandwich ELISA method was established for detecting BHV-1. The ascites2G4with indirect ELISA titers of128000was used for capture antibody, the rabbit polyclonal antibody against BHV-1gB was used for the second antibody. Phalanx test determined that the optimal concentration of capture antibody was4μg/mL; optimal working concentration for the rabbit polyclonal antibody against BHV-1gB was65ng/mL; and minimum detection quantity for BHV-1was80ng/mL. Twenty samples were tested by the double-antibody ELISA method,8of them were positive, and coincidence rate was90.0%comparing with polymerase chain reaction (PCR) amplification results, indicating that the double-antibody sandwich ELISA can be used in the clinical diagnosis of BHV-1infection.