| Infectious bovine rhinotracheitis(IBR) is an acute,febrile, and contact infectious disease which was caused by infectious bovine rhinotracheitis virus(IBRV). IBRV is able to infect a lot of tissues and cause various clinical syndromes such as rhinotracheitis, pustular vulvovaginitis and balanoposthitis, abortion, infertility, conjunctivitis and encephalitis in bovine species. Latent infection established by IBRV in the rigeminal ganglion would result in persistent infection and virus shedding, which makes it difficulty to eradicate completely. Secondary fatal bacterial pneumonia from acute IBR is one of the most important causes of economic loss in the cattle industry. IBR was first found in American in 1950 s, then transmitted all over the world. Serological investigation showed that IBR infection existed in most parts in China. g D, one of the major glycoproteins settled in the envelope of IBRV, plays an important role in the process of adsorption and invasion and is capable of inducing neutralizing antibody. There is no standardized IBRV detecting method until now in China, so it is necessary to do some research on the related detection methods.In this study, g D of IBRV was expressed in E. coli. The immunogenicity of recombinant protein was testified with Western blot analysis. 13 MAbs were obtained after conventional cell fusion between sp2/0 myeloma cells and spleen cells from 6-8 week-old BALB/c mice immunized with purified IBRV for three times and recombinant g D for one time. Four of the MAbs(2D8, 3F9, 1G3, and 5B7) were reactive with both the recombinant g D and IBRV,while the others(2E4, 3C2, 4E8, 1F5, 4H5, 6B6, 3F3, 2A9, and 4D11) were reactive only with IBRV. The indirect immunofluorescence assay(IFA) and Western blot analysis demonstrated that all the MAbs possess satisfying reactivity, while specificity test showed that the MAbs were reactive only with IBRV, not with bovine adenovirus type 3(BAV-3) and bovine parainfluenza virus type 3(BPIV3). The neutralization titer of the ascites of 1G3 was 1:32 deternined by virus neutralization test.The antigen epitope of MAb 1G3 was investigated by pepscan method with GST-fused peptides based on MAb and a linear epitope 259EESKGYE265 was identified as the smallest unit of maximum reaction activity of the epitope, and 260ESKGYE265 was identified as the minimum activity unit. The amino acid sequence alignment of the g D of IBRV(also called as bovine herpesvirus 1(BHV-1)) showed that the epitope is completely conserved among different isolates of BHV-1, while there is a great difference in some ruminant alphaherpesviruses related to IBRV such as caprine herpesvirus 1(Cp HV-1), cervid herpesvirus 1(Cv HV-1), and elk herpesvirus 1(El KHV-1).Meanwhile, a double antibody sandwich(DAS) ELISA was established with MAb 3C2 as coating antibody and horseradish perioxdase(HRP) conjugated MAb 1F5 as detecting antibody for detection of IBRV. Specificity test showed that the DAS-ELISA could detect IBRV, but could not detect BAV-3, BPIV3, and bovine viral diarrhea virus(BVDV). The cut-off value was determined as 0.24 according to the detection results of 24 negative samples for IBRV. When OD value measured at 450 nm was great than 0.24, the sample was identified as positive; otherwise the sample would be regarded as negative. The sensibility test of the DAS-ELISA showed that the minimum detection is 102.9TCID50/0.1ml. Repeatability test indicated that the DAS-ELISA possessed favorable repeatability.In summary, 13 MAbs against IBRV were obtained in the study. An antigen epitope of MAb 1G3 was identified. A DAS-ELISA that was able to detect IBRV was established. The DAS-ELISA might be used for detection of IBRV. This study would be useful for studying biological structure and function of the g D of IBRV and for studying the pathogenesis of IBRV, and would provide technical support for the pathogen diagnostics of IBRV. |
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