Preparation Of Monoclonal Antibody Against GD Protein Of Infectious Bovine Rhinotracheitis Virus And Establishment Of Indirect Immunofluorescence Assay

Infectious bovine rhinotracheitis(IBR)is an acute contact infectious disease in cattle caused by Infectious bovine rhinotracheitis virus(IBRV).It is clinically characterized by high fever,rhinitis,and upper respiratory tract inflammation,and can cause Infectious pustular vulvovaginitis,balanitis,milk loss of cows,and abortion.After IBRV infection,a latent infection can be established in the nervous system to form a persistent infection.The diseased cow has been infected for a long time or even for life,making it difficult to eradicate.The virus was isolated from dairy cows imported from New Zealand in1980 for the first time in China.Antibody-positive cattle have been detected in various regions since then,and the virus has also been isolated from imported cows with no clinical manifestations.Serological surveys show that in addition to Hong Kong,Macau,Taiwan,and Jiangxi provinces,the cattle in other administrative provinces,cities,or autonomous regions have been infected with IBRV,indicating that IBR has been widely spread throughout our country,which has brought great economic loss to our cattle industry.The gD protein is necessary for IBRV to bind to cells,and can induce humoral and cellular immunity.Monoclonal antibodies against gD protein have the ability to neutralize viruses.Therefore,gD protein is an important protein for the development of diagnostic reagents or antiviral research.In this study,IBRV gD protein expressed by CHO cells was used as an immunogen to immunize8-week-old BALB / c mice.The spleen cells and SP2 / 0 cells were aseptically taken for cell fusion,and three hybridoma cell lines were screened which were named 4G3D4,6E8B7,9D7A7.And one of the monoclonal antibodies with the highest antibody titer,namely 4G3D4,was selected for subsequent experiments.The purified concentration of 4G3D4 was 2.6 mg / mL;the antibody subclass was IgG1;and it could react with IBRV and produce specific fluorescence with MDBK cells inoculated with IBRV;the affinity constant,indirect ELISA,and indirect immunofluorescence assay(IFA)all showed that the hybridoma cells secreting the monoclonal antibody 4G3D4 had good genetic stability.Using the E.coli pET-28 a prokaryotic expression system,truncated expression of IBRV gD protein,identification the epitope recognized by mAb 4G3D4,and Western Blot showed that the epitope targeted by mAb 4G3D4 was a linear epitope,which was located in the amino acid region of315aa-348 aa of the gD protein.Analysis of the epitope conservation showed that the epitope was relatively conserved among the isolates of IBRV.At the same time,the IBRV gD protein monoclonal antibody 4G3D4 prepared in this study was used as the primary antibody,and anti-mouse IgG-FITC antibody produced by goat was used as the secondary antibody.By optimizing each reaction condition,an indirect immunofluorescence assay(IFA)was established.By comparing with the IBRV gD protein monoclonal antibody imported from the United States VMRD company,different isolates of IBRV,Bovine viral diarrhea virus(BVDV),Bovine Parainfluenza Virus Type 3(BPIV3),were detected.The results were consistent,and the coincidencerate was 100%.The results of intra-and inter-assay repeatability tests showed that the method had good repeatability.The results of specificity test and sensitivity test showed that indirect immunofluorescence assay had high sensitivity and strong specificity,and was suitable for clinical detection of IBRV antigen.In summary,three hybridoma cell lines were screened out in this study.And one of the monoclonal antibodies with the highest antibody titer,namely 4G3D4,was selected for subsequent experiments.The monoclonal antibody had strong stability and good reactivity with IBRV.Analysis of its epitope revealed an antigenic epitope.At the same time,the prepared IBRV gD protein monoclonal antibody4G3D4 was used as a primary antibody,and an indirect immunofluorescence assay for detecting IBRV was initially established for the detection of IBRV antigen in bovine and bovine-derived biological materials.This study laid the foundation for the indepth study of the biological structure of IBRV gD protein and the pathogenicity of IBRV,and also provided technical support for the diagnosis and prevention of IBRV.