Prokaryotic Expression Of Bovine Infectious Rhinotracheitis Virus GE Protein And Preliminary Establishment Of Indirect ELISA Detection Method

Bovine infectious rhinotracheitis virus(IBRV),also known as bovine herpes disease type 1(BHV-1),mainly causes an acute,thermal,and contact infectious disease of cattle,mainly showing high fever,difficulty breathing,rhinitis and Inflammation of the respiratory tract has brought great economic losses to the cattle industry.Bovine contagious rhinotracheitis is rapidly prevalent in Europe and difficult to control.The main purpose of this test is to establish an indirect ELISA detection method for detecting IBRV antibody by using the prokaryotic expression product of bovine infectious rhinotracheitis ge gene as an antigen.The primer sequence of IBRV gE gene was designed by biological software,and then the gene was amplified by PCR,and the Pcold-TF-gE prokaryotic expression system was constructed.Recombinant protein gE is induced and expressed in supernatant form.Westen blot detection showed that the recombinant expression product could be specifically recognized by IBRV standard positive sera.The purified and identified expression product was used as antigen to coat the microplate with 1 ?g/mL,HRP rabbit anti-bovine was used as the secondary antibody,and the TMB color development time was 15min.The results showed that the cut-off value was 0.369,and the specific test showed that the method did not cross-react with positive sera of foot-and-mouth disease,bovine viral diarrhea,paratuberculosis,brucella,and mycoplasma.The results of the sensitivity test showed that the dilution ratio of bovine positive sera reached 1:4096 indicating good sensitivity.The results of inter-plate and in-plate tests on 5 copies of IBRV positive sera showed good repeatability.Using the established gE-ELISA method,at the same time,compared with IDEXX(IBR)antibody detection kit for 200 clinically positive sera,the coincidence rate reached 95%.Therefore,this method is particularly sensitive and efficient,and can be used for the detection of bovine infectious rhinotracheitis serum antibodies,laying the foundation for the IBRV diagnostic kit.