Establishment Of An Indirect ELISA Assay For Bovine Respiratory Syncytial Virus Nucleoprotein Based On Insect Baculovirus System

Bovine respiratory syncytial virus（BRSV）is an enveloped,single-stranded,negative-stranded RNA virus.It is a member of the Paramyxoviridae family,subfamily Pneumoviridae,genus Pneumovirus,and is one of the main agents of the respiratory disease complex（BRDC）in dairy cattle and beef calves.BRSV can be transmitted by direct contact or aerosol,causing severe respiratory disease with clinical manifestations such as fever,runny nose,cough and,in severe cases,death.Currently,the main diagnostic methods for BRSV are haemagglutination inhibition assay（HI）,neutralisation assay（NT）,enzyme-linked immunosorbent assay（ELISA）,reverse transcription polymerase chain reaction（RT-PCR）and quantitative real-time fluorescence PCR（q RT-PCR）.Among them,ELISA methods have the advantages of simplicity,rapidity and sensitivity,but there are no commercial ELISA kits in China yet,while imported ELISA kits are expensive,which seriously hinders the prevention and control of BRSV in China.Therefore,the establishment of an ELISA method for the detection of BRSV is of great significance for the epidemiological investigation,monitoring as well as prevention and control of the disease.The N protein of BRSV is a very important structural protein among all proteins and has good immunogenicity.In this study,a baculovirus expression vector was constructed using the N gene sequence of BRSV,and the expressed N protein was used to establish an indirect ELISA method for detecting BRSV.In this study,a pair of primers for complete amplification of the N gene sequence was designed based on the N gene sequence of the BRSV reference sequence on Gen Bank（accession number:NC001989.1）,and the Bam HⅠand XhoⅠrestriction endonuclease sites were introduced in its 5’,respectively.The N gene sequence was amplified by PCR and double cleaved,and the N gene sequence was ligated to the transfer vector p Fast Bac-HTB by T4 ligase,then transposed to the baculovirus vector by DH10Bac receptor E.coli,and finally the recombinant baculovirus Bacmid-BRSV-N was constructed by transfecting Sf9 insect cells,which could stably express the recombinant protein.SDS-PAGE and Western-blot analysis indicated that the recombinant protein was expressed in the form of secreted in supernatant.The protein was purified by His-Ni affinity chromatography using culture medium supernatant,and Western-blot reaction was performed with the recombinant protein using BRSV-positive serum as primary antibody.The results showed that the recombinant BRSV N protein could react specifically with BRSV-positive sera.Using the purified recombinant BRSV N protein as the encapsulated antigen and BRSV-positive serum as the primary antibody,an indirect ELISA diagnostic method was established.The optimal reaction conditions for the indirect ELISA were determined through a series of optimisation:antigen encapsulation concentration of 8μg/m L,overnight encapsulation at 4℃,800-fold dilution of the serum to be tested,1h closure with 5%skimmed milk powder,2h reaction time for the antigen and primary antibody（serum to be tested）,5000-fold dilution of HRP-labelled secondary antibody,1.5h reaction,20min TMB colour development.Thirty BRSV negative sera were tested to calculate the threshold value,and it was determined that when the OD₄₅₀ of the serum was greater than 0.215,it was positive,and when the OD₄₅₀ was less than 0.178,it was negative.Samples whose OD₄₅₀ was between the negative and positive threshold values were considered suspicious and needed to be re-tested.The indirect ELISA method developed in this experiment was tested for positive sera for bovine infectious rhinotracheitis virus（IBR）,bovine parainfluenza virus type 3（BPIV-3）,bovine viral diarrhoea virus（BVDV）,bovine rotavirus（BRV）and bovine coronavirus（BCV）,and the results were all negative,indicating that the method has good specificity.BRSV-positive sera of bovine origin were still positive at 1280-fold dilution,demonstrating the good sensitivity of the method.The results of the intra-batch and inter-batch reproducibility tests were within 10%of each other,demonstrating that the method is also reproducible.Using the established ELISA assay and commercial ELISA test kits and testing 200 bovine sera from some cattle farms in Jilin Province,the compliance rate between the two was 88.5%,while the BRSV antibody positivity rate was found to be over 30%in some cattle farms in Jilin Province.In summary,this study successfully established an indirect ELISA method for the detection of BRSV and demonstrated that the established indirect ELISA method can be used for the detection of samples to provide a basis for the detection of BRSV,...