Establishment And Application Of New Detection Methods Based On GG Gene For Bovine Infectious Rhinotracheitis Virus

Infectious bovine rhinotracheitis (IBR) is caused by infectious bovine rrhinotracheitis viruses (IBRV) and known mainly as an acute, pyrexic and contagious disease of respiratory tract which is charaeterized by rhinotracheitis, conjunctivitis, fever, abortion and infectious pustular vulvovaginitis. Therefore, this study is to develop monoclonal antibodies for infectious bovine rhinotracheitis virus gG protein, the infectious bovine rhinotracheitis virus gG gene missing restructuring virus detection methods, finally provide matching detection method which can differentiate wild virus and vaccine. This research designed the primers specific to IBRV-gG gene and established polymerase chain reaction(PCR) to detect and differiate IBRV from other members of herpesviruses. Furthermore, the truncated IBRV-gG gene was cloned, expressed and purified., with the recombinant IBRV-gG protein was used to develop monoclonal antibodies and polyclonal antibodies. The ELISA to detect IBRV-gG or its antibody was established. The main results of this paper were summarized as follows:1. Establishment of a PCR to detect gG gene of Infectious Bovine Rhinotracheitis VirusA PCR method was established successfully to detect Infectious Bovine Rhinotracheitis Virus (IBRV) and differentiate bovine herpesviruses. The sensitivity for IBRV was 0.002 PFU/ml. Since this PCR has good sensitivity and specificity, it would have a wide application in diagnosis of bovine herpesvirus infection and differential detection among the members of herpesviruses and between natural infection and vaccination of gG deleted marker vaccine.2. The cloning of Infectious Bovine Rhinotracheitis Virus truncated glycoprotein G and expression in E.coliAccording to published IBRV gG gene sequences in GenBank (GenBank accession number NC₀01847.1), gG gene which does not contain succoth and the watershed signal peptide was amplified and cloned into pMD18-T. The fragment was futher subcloned into the expression vector pET-32a. The SDS-PAGE and Western blot analysis indicates that the truncated gG gene in E. coli BL21(DE3) was expressed in both soluble and inclusion body forms, and the recombinant protein had immunological activities.3. Preparation of monoclonal antibody and polyclonal antibody to infectious bovine rhinotracheitis virus gG proteinThe purified IBRV and recombinant proteins gG were immunized in Balb/c mice. The SP2/0 cells and the splenic cells from the immunized mice was fused. The hybridoma cells were screened with purified IBRV wild strain and the IBRV-ΔgG mutant in parallel with indirect ELISA screening. The positive hybridoma cells were cloned by limited dilution method for three times and the resultant monoclonal antibodies (McAb), which was obtained. The Western blot assay confirmed that it specifically reacted with the wildtype IBRV but not IBRV-ΔgG mutant. ELISA titer of the McAb mouse ascites reached 51200. This monoclonal antibody can be used to IBRV detection, and provide tools for infectious bovine rhinotracheitis eradication program.4. Establishment of indirect ELISA to detect infectious bovine rhinotracheitis IBRV-gG proteinThe gG sandwich ELISA to detect IBRV gG was established. Briefly, McAb 3D6 was coated onto the ELISA plates, while rabbit polyclonal antibody to gG was overlaid as the capture antibody.And take this method to detect IBRV,BT,BM, BVDV-2,IBRV,BRSV and PIV-3, The result is said that this method is very sensitive.5. Establishment of competitive ELISA to detect infectious bovine rhinotracheitis IBRV-gG antibodyBy use the purified recombinant gG protein as the coating antigen, horseradish peroxidase (HRP) labelled monoclonal antibody 3D6 as the competitive antibody to establish the competitive ELISA to detect antibody to gG. The gG antigen was coated at concentration of 500ng/mL, serum dilution was 1:160, HRP-3D6 dilution was 1:51 200, and more than 50% of inhibition rate was determined as positive.