Establishment And Preliminary Application Of Indirect ELISA Method For Detection Of Bovine Coronavirus Antibodies Based On N Protein

Bovine coronavirus（BCoV）is an important pathogen causing calf diarrhea（CD）,adult bovine winter dysentery（WD）and bovine respiratory disease（BRD）of different ages.Calves show severe diarrhea and even death when infected,and adult cattle show a significant reduction in milk production when infected.In recent years,bovine coronavirus has spread widely around the world,which has had a serious impact on the development of the cattle industry around the world.At present,there is no specific treatment drug for bovine coronavirus infection,it is important to detect sick animals in time and take appropriate measures.Due to the lack of effective diagnostic techniques in clinical practice,therefore,this experiment established an indirect ELISA method for the detection of bovine coronavirus antibodies through the study of bovine coronavirus N protein,and 296 clinically collected cow serum samples were preliminarily detected.The result are as follows:1.The S gene,N gene,HE gene,M gene and E gene of the Shaanxi isolate of bovine coronavirus were successfully cloned.The cloned S gene has 97.5%～99.3%homology with26 reference strains,which is the closest genetic distance to Chinese strains SWUN/NMG-D10/2020 and CH/HB-BD/2019;The cloned N gene had 97.6%～99.5%homology with 24 reference strains,which is the closest genetic distance to the Chinese strain CH/GS-1/2019;The cloned HE gene had 95.8%～98.9%homology with 24 reference strains,which is the closest genetic distance to the Chinese strains SWUN/B9/2018 and SWUN/B6/2018;The cloned M gene had 97.8%～99.9%homology with 22 reference strains,there was no significant genetic difference with the reference strain;The cloned M gene had99.1%～100%homology with 18 reference strains,there was no significant genetic difference with the reference strain.2.The prokaryotic expression recombinant plasmid pET-28a-N was successfully constructed.Through the optimization of the induction conditions,the final concentration of1 mmol/L IPTG was selected for induction at 37°C for 6 hours.After purification by Ni column,high-purity and high-concentration N protein was obtained with protein size of 50 ku.After identification by Western blot,the recombinant N protein has good reactogenicity.3.An indirect ELISA detection method for bovine coronavirus antibody based on N protein was successfully established.The amount of antigen coating was 1.5μg/m L,the blocking solution was 5%goat serum,and the reaction was performed for 120 minutes.The serum dilution was 1:50,and the reaction was performed for 90 minutes.The HRP Goat-Anti-Bovine Ig G dilution was 1:10000,and the reaction was performed for 60 minutes,and the color was developed by TMB for 10 minutes.The critical OD_450nm value of negative and positive was 0.256.The method has good specificity,sensitivity and reproducibility.The bovine coronavirus antibody indirect ELISA detection method established in this experiment was used to detect 296 dairy cow serum samples collected clinically,of which 54 were positive samples,and the positive rate was 18.24%.In summary,the S gene,N gene,HE gene,M gene and E gene of shaanxi epidemic bovine coronavirus strain were cloned and sequence analysis in this study.Secondly,the prokaryotic expression recombinant plasmid was constructed,and an indirect ELISA method for detecting bovine coronavirus antibody using N protein as the coated antigen was established to provide technical support for the laboratory diagnosis of bovine coronavirus.