Isolation And Identification Of Infectious Bovine Rhinotracheitis Virus And Preparation Of Monoclonal Antibody

In the mid April of 2006, a bovine disease, likely due to infectious bovine rhinotracheitis (IBRV) infection was widely spread in cattle herds of Daqing city, Heilongjiang province. To isolate IBRV, samples were collected from vaginal secretions of sick cattle with high fever. The samples were cultured in MDBK cell line and continuously cultured for 3 passags with significant cytopathic effect. The culture solution of MDBK cell was collected and virus particals were purified by differential ultracentrifugation.The viral particals were concentrated 320 fold and the agglutination titer was 1:1280 for the murine erythrocytes. The viruses were further confirmed as IBRV since they could be neutralized with positive bovine sera of IBRV with the neutralization titer of l:27. The conservative region of gB gene of IBRV was amplified with a pair of specific primers. The result of PCR showed that the size of gB gene was 298 bp. The agreement of gB gene sequence between the isolated viruses and IBRV M14106 from GenBank was 99.3%. The gD gene of IBRV was also cloned and the sequenced. The homology of amino acid and nucleotide sequences of gD gene were 97.6%～98.2%, 97.1%～97.6%, respectively, in comparison with AJ004801, M59846, Z23068 and Z98199 of IBRV strains in GenBank. Phylogenetic tree of gD gene was then contructed. Furthermore, the physical and chemical natures of the isolated IBRV were determined as they are sensitive for diethyl ether, chloroform, acid and trysin digestion. They were completely inactivated at 50℃within 1h and not protected by Mg2+.The 6-week-old female Balb/c mice (body weight for 18-22g) were immunized intraperitoneally for 4 times with the isolated IBRV which was purified and concentrated by sucrose density gradient centrifugation. The spleen cells of immunized mice were fused with SP2/0 myeloma cells. Indirect enzyme linked immunosorbent assay (ELISA) was used to screen hybridomas. The positive clones were subcloned 3 times by limited dilution. Three monoclonal antibodies (McAbs) (designated 3A1,3D1 and 5D2) were specific against IBRV with the subtype of IgG1κand the titers of 3 McAbs substrates were: 1:3200, 1:3200, 1:6400 respectively. The titers of ascites were: 1:128000, 1:16000, 1:256000 respectively. The result of western blot indicated that it was specific to bind IBRV. The specificity assay result illustrated it had no cross reaction with bovine coronavirus, bovine rotavirus, bovine adenovirus and bovine respiratory syncytial virus. The results of these experiments confirmed that the isolated virus was infectious bovine rhinotracheitis virus and named as IBRV DQ01 strain. It was proved that we have developped hybridoma cell lines which serected monoclonal antiboies against infectious bovine rhinotracheitis virus.