Prokaryotic Expression Of Bovine Viral Diarrhea Virus E2 Protein And Establishment Of An Indirect ELISA Method

Bovine viral diarrhea virus(BVDV) can cause a complex,multi-clinical disease in cattle.The main symptoms are diarrhea,acute and chronic mucosal disease,congenital defects,immunosuppression,immune tolerance,and persistence.Infection,abortion of female animals,stillbirth and deformed tires,etc.The infectious diseases caused by this infection have seriously affected the development of the world cattle industry.With the large-scale cultivation and the frequent transportation of breeding stocks,the trend of the disease in China has gradually increased,which has brought huge economic losses to the cattle industry.Therefore,this study used nasal swab samples and blood samples collected from some pastures around Inner Mongolia.After treatment,the samples were inoculated into MDBK cell culture flasks that had grown into a single layer,and were identified as positive by PCR with the designed primers.The bovine viral diarrhea virus was isolated and the purified virus was used for subsequent experiments.First,we analyzed the major coding region of bovine viral diarrhea virus(BVDV-1)E2 protein.The BVDV-1 E2 gene was cloned into the expression vector Pcold-TF by PCR amplification technology,and Pold-TF-E2 was constructed.The cells were transformed into competent cells BL21,and the induced expression of the recombinant protein was carried out using IPTG,and the conditions for inducing expression were 0.8 mMol/L IPTG induction at 37? for 4 h.The collected bacterial solution was lysed by sonication,purified by Ni-IDA protein purification kit and identified by SDS-PAGE and Western blot.The expressed recombinant protein E2 was identified as 82 Kda,which was consistent with the expected band.An indirect ELISA method for detecting bovine viral diarrhea virus was established by using the cleavage-expressed product E2 protein as a coating antigen.The optimal conditions for determining the reaction were as follows:the antigen coating concentration was 8 ?g/mL,the optimal temperature and time for coating was 4? for 16 h,the primary antiserum dilution ratio was 1:100,and the reaction temperature and time were 37? for 1 h.The best blocking solution is 10%PEG4000,blocked at 37? for 1 h,the dilution of the enzyme-labeled secondary antibody is 1:6000,and the reaction temperature and time are 37? for 1 h.The critical value of the yin and yang determination was determined by experiments to be 0.33.The method has no cross-reaction with positive sera such as bovine infectious rhinotracheitis,bovine infectious pleuropneumonia,red feather disease,paratuberculosis,brucellosis,and small ruminant plague,indicating that the method has good specificity and will be BVDV.The positive serum was judged to be positive when diluted to 1:4096,indicating that the established method has high sensitivity,and the coefficient of variation of the repeated test between groups and groups is less than 5%,indicating that the method has good repeatability.189 samples of serum were tested for clinical samples using the indirect ELISA method established above and the method established by the kit of IDEXX.The coincidence rate of the two was 92.8%.From the above results,the indirect ELISA method established in this experiment can provide a convenient and powerful means for rapid diagnosis and epidemiological investigation of the disease,and the cost is low.