Establish Indirect ELISA Using Antibody For Bovine Parainfluenza Virus Type 3 With Recombinant NP Protein As Antigen

Bovine Parainfluenza virus Type 3（BPIV3）is a kind of Bovine respiratory disease complex（BRDC）which can stimulate the development of immune suppression.BRDC is the main pathogen of bovine respiratory disease,which causes great economic loss to cattle industry every year.According to the results of epidemiological investigation,BPIV3 is widely prevalent in cattle in China,and there is no specific drug treatment at present.At the present stage,the detection method of BPIV3 in China is still in its infancy,and there is no sensitive and accurate commercial detection kit independently developed.BPIV3 can be transmitted by aerosol,and has a strong transmission power and secondary harm.Therefore,it is more important to establish an economical,rapid and effective detection method for BPIV3.In this study,the full sequence of NP protein was amplified by RT-PCR method,evolutionary relationship analysis and antigenic epitope screening were performed after sequencing.The analysis showed that NP had high genetic stability and good antigenicity.Ligated to the p ET-30a vector,cloned into p ET-30a.The recombinant plasmid p ET-30a-NP was transformed into Escherichia coli for induced expression.SDS-page showed that the recombinant NP protein was expressed as an inclusion body with a size of 66 k Da.BPIV3 antibody indirect ELISA was established using NP protein as coated antigen.By optimizing the ELISA reaction conditions and comparing the P/N values,the following reaction conditions were selected:antigen coating concentration of 4μg/m L,incubation at 37℃for 1 h,coating overnight at 4℃;PBST containing 2%BSA was sealed at 37℃for 2 h.The dilution of primary antibody serum was 50 times,and incubated at 37℃for 1 h.The enzyme-conjugate secondary antibody was diluted 5000 times and incubated at 37℃for 1 h.TMB was displayed for 15 min,and OD4₅₀nm was determined with a microplate reader after termination.The criteria of the ELISA method are as follows:S/P value≥0.187 is considered as positive,and S/P value<0.161 is considered as negative.The method still maintained good sensitivity when the positive serum was diluted 160 times.The coefficient of variation of both intraplate and interplate repeatability tests was less than 8%.The positive sera of bovine brucellosis,FMD,and bovine paratuberculosis were detected,and the results were all negative,which proved that the established method did not have serological cross-reaction with the above viruses and was highly specific.The ELISA method was compared with neutralization test,and the coincidence rate was 84.5%.Using the established BPIV3 antibody indirect ELISA to detect the antibody-positive rate of bovine serum samples from 4 large-scale ranches in Heilongjiang Province from 2009 to 2013,the antibody positive rate was 28.13-54.17%（44.92%）,and the positive rate of antibodies was28.13-54.17%（44.92%）in 29 large-scale ranches in parts of Northeast China in 2021.The antibody-positive rate of bovine serum samples was 36.36-83.33%（63%）,and BPIV3 infection was found in all tested pastures.