Isolation And Identification Of Bovine Respiratory Syncytial Virus And Establishment Of An Indirect ELISA Diagnostic Method With The Recombinant Nucleocapsid Protein

Specific primers were designed and synthesized according to the reported nucleocapsid (N) protein gene sequence from bovine respiratory syncytial virus (BRSV) in GenBankTM. The size of 596bp fragment was amplified by different condition optimization, then the RT-PCR diagnostic method of BRSV was constructed by the specific, sensitive and reproducible assays. The sensitivity of this method could detect at least 1 TCID50/mL. The diagnostic method of BRSV was no cross-reaction with the closely related clinical symptom pathogen, such as bovine adenovirus, bovine parainfluenza virus, bovine infectious rhinotracheitis virus, Anaplasma margina. The results of a series of tests indicated that it was a rapid, sensitive, specific, reproducible method, and could be used as a method of detection for bovine respiratory syncytial virus infection.A bovine disease, likely due to infectious BRSV was widely spread from a beef farm in Heilongjiang province. In order to isolate BRSV, clinical samples were collected from nasal secretions of an invasive bovine. The samples were inoculated to Vero cells, and one virus strain was successfully isolated. The isolated virus could produce stable typical syncytioma cytopathic effect in Vero cells. The isolated virus was insensitive to 5-iodo-2-deoxyuridine, but sensitive to ether, chloroform, trypsin and acid, it was not resistant to heat, it could not be protected by Mg2+, and it could not be agglutinated and adsorbed erythrocytes of human, chicken and some animals by physicochemical experiments. The virus could be neutralized by BRSV standard positive serum. The RNA extracted from the cell culture of the isolated virus was subjected to RT-PCR analysis, utilizing a pair of specific primers, then the sequence, which consisted of 596bp lying in the gene encoded the nucleocapsid protein was amplified. The nucleotide identity of the fragments of the N genes between BRSV strains in GenBankTM database and the isolate ranged from 97.8% to 99.3%. The results showed that the isolated virus was bovine respiratory syncytial virus, then it was named BRSV HJ strain.Total RNA was extracted from the supernatant of cell culture. The gene of N protein was amplified by RT-PCR from the total RNA, then cloned into pMD18-T vector. The positive plasmid pMD18-T-N was constructed by screening and identification. Then the gene of N protein was subcloned into the downstream of the pET-30a (+) vector, which was named as pET-30a-N. It has been confirmed that the target gene was inserted into the pET-30a (+) vector by PCR and restriction enzyme digestion. It was proved that the recombinant plasmid pET-30a-N contained the gene of N protein, which had correct sequence by sequence analysis. The nucleotide identity of the N genes between BRSV strains in GenBankTM database and the isolate ranged from 97.5% to 99.1%, the amino acid identity was ranged from 98.7% to 99.2%. The recombinant strain E. coli BL21 (pET-30a-N) was induced by 1mmol/L IPTG. The recombinant N protein was expressed at high level. It has been proved that the recombinant N protein of BRSV HJ strain has good reactionogenicity by western blot. Taking the purified recombinant N protein as diagnostic antigen, it was established that indirect ELISA method for detecting BRSV antibody. The optimal reaction conditions of indirect ELISA were determined by experiments. The best condition of ELISA: the optimal coating buffer was carbonate buffer solution (50mM pH9.6), and the optimal coating antigen for microplate was 5.0μg/mL. Five percent skim milk was added as the optimal blocking agent and incubated for 1 hour at 37℃. The best serum dilution buffer was 5% skim milk, and the optimal dilution of sera samples was 1:100. The optimal working concentration of HRP-labeled rabbit anti-bovine IgG was 1:5 000. The results showed that there is 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 some area in Heilongjiang province were tested by developed indirect ELISA method and the positive rate is 27.33%.It has been proved that some area in Heilongjing province exist bovine respiratory syncytial disease by the assay. It is very important that development of indirect ELISA for the preparation of ELISA kit. It is useful for the study of epidemiology, immunology, pathogenesis, immunoprophylaxis and other aspects of bovine respiratory syncytial disease.