Preparation Of Monoclonal Antibody Against Nucleoprotein Of Bovine Respiratory Syncytial Virus, And Experimental Infection Of Guinea Pig With The Virus

Bovine respiratory syncytial virus (BRSV), which is a negative-stranded RNA virus, belongs to tothe pneumovirus genus within the family Paramyxoviridae. BRSV is a major cause which contributes tothe respiratory disease of the young calves.The infectivity rates of BRSV are always rather high, but themortality is low. However, with the rapid development of livestock, the mortality caused by BRSV isincreasing. BRSV outbreaks always occur during winter which mostly infects the young calves.Because BRSV is not in the list of quarantine, the infected calves may be introduced into our countrywhich result in the spread of BRSV. The study of BRSV is needed urgently to control this virus.The nucleoprotein gene was amplified by RT-PCR from RNA of BRSV and cloned into pET30a.The transformed BL21(DE3) with pET30a-N were cultivated and induced with IPTG. The nucleoprotein(N) was successfully expressed in E.coli BL21with a molecular weight of approximately49kD andanalyzed with Western blotting. To prepare monolconal antibody (MAb) to nucleoprotein of BRSV,BALB/c mice were immunized with purified recombinant N (rN) expressed in E.coli and twohybridomas secreting MAb were obtained by screening from the SP2/0cells fused with the spleen cellsof the immunized BALB/c mice by indirect ELISA coated with BRSV. The titers of MAbs2D12and4B10in ascites were1×10~5,1×10~6and1×10~2,1×10~3as detected by rN and BRSV itself, respectively.The MAbs secreted by hybridomas2D12and4B10had highly reactivity and specificity in indirectELISA, western blotting and IFA, and identified as IgG1with a light chain of κ by indirect ELISA. Thespecific tests indicated that the MAbs2D12and4B10had no reaction with bovine parainfluenza virustype3and bovine viral diarrhea virus. Therefore, the MAbs2D12and4B10could be used to establishdiagnosis method for BRSV.To study the replication of BRSV in guinea pig, experimental infection with BRSV strain375wasconducted. The replication of virus and pathological changes were evaluated.The distribution of BRSVwas examined by RT-nested-PCR and results showed that BRSV can be detected in24hours afterinoculation. BRSV can be detected in almost all the viscera, and the result showed that BRSV canreplicate in the blood and transmit to the organs of guinea pig. The hispathologic examinations showedthat interstitial pneumonia was present. The results of immunofluorescence staining revealed that BRSVcan replicate in the organs of guinea pig. Thus this experiment lays the root for studying of laborotoryanimal model of BRSV.