Development of an indirect ELISA diagnostic method for detecting bovine respiratory syncytial virus using recombinant nucleocapsid protein and its preliminary application.

【Objective】The ELISA plate was coated with the purified bovine respiratory syncytial virus(BRSV) HJ strain recombinant nucleocapsid(N) antigen and the optimal reaction conditions of indirect ELISA were determined by experiments.The indirect ELISA method for detecting BRSV antibody was established by determining the cut off value.【Method】The recombinant E.coli strain BL21 containing the recombinant plasmid pET-30a-N was induced by IPTG.The recombinant N protein was expressed at high level,then it was purified by electroelution method.The recombinant N protein was identified by western blot.Taking the purified recombinant N protein as diagnostic antigen,the indirect ELISA method for detecting BRSV antibody was established. 【Result】The recombinant N protein was successfully expressed and purified,it was proved that the recombinant protein has good reactogenicity by western blot.Taking the purified recombinant N protein as diagnostic antigen,the indirect ELISA method for detecting BRSV antibody was established.The results showed that there was no cross reaction between the recombinant N protein and the positive serum of Anaplasmosis,IBRV,BPIV.Compared with the serum neutralization testing,the results indicated that the specificity,sensitivity and coincidence of the developed indirect ELISA was 85.0%,90.9%and 87.7%,respectively.A total of 600 sera of cattle,which obtained from Mudanjiang,Jamusi,Hegang,Daqing in Heilongjiang province,were tested by the developedindirect ELISA method and the positive rate was 27.33%.【Conclusion】The developed indirect ELISA has good specificity,sensitivity and reproducibility,it has been proved that BRSV is existed in some areas in Heilongjing province.It is very important that the development of indirect ELISA and its preliminary application of BRSV in cattle for the preparation of ELISA kit,diagnosis and epidemiological investigation of bovine respiratory syncytial disease.