| Bovine viral diarrhea/mucosal disease(BVD/MD) is caused by the infection of cattle with bovine viral diarrhea virus(BVDV).BVDV infection is characterized by reproductive failure,congenital abnormalities and general immunodepression to unthrifty calves and severe mucosal disease.BVDV is grouped in the genus Pestivirus within the family of Flaviridae together with other important pathogens, such as classical swine fever (CSFV) and border disease virus(BDV). BVD is spread worldwide and goes along with high economic losses in cattle industry. And so does it in China. Therefore it is of significance to come up with a reliable diagnosis method.Erns is also called as EO and is one of the most conversed glycoprotein of BVDV and it can Induce the production of neutralizing antibody. A pair of specific primers was designed based on the genome sequence of VEDEVAC strain. A fragment of about680bp was amplified by RT-PCR, and the target fragment was cloned into the pET-30a vector. The positive recombinant plasmids were obtained by enzyme digestions and sequenced. The identified recombinant plasmid was transformed into E coli BL21. The recombinant protein EO was expressed in inclusion body form after incubation of transformed E coli BL21and induction with IPTG. The recombinant EO protein was analyzed by Western blotting and it had a good antigenicity. BALB/c mice were immunized with purified recombinant EO expressed in E.coli and four hybridomas secreting monoclonal antibody(MAbs) were obtained from fusing the SP2/0cells with the spleen cells of the immunized BALB/c mice by an in direct ELISA coating with BVDV. The titers of MAbs1E6,3C3,3F4,3F7in ascites were4×104,3.2×105,3.2×105,2×104respectively, as detected by indirect ELISA. The MAbs secreted by hybridomas1E6,3C3,3F4,3F7had highly reactivity and specificity in indirect ELISA,Western blotting and immunofluorescence staining, and identified as IgGl with a light chain of λ by indirect ELISA The purified recombinant EO protein was also used as immunogen against for immunizing rabbit to obtain polyclonal antibody(PAb) against the recombinant EO protein. The PAb against the recombinant EO protein also has good highly reactivity and specialty in immunofluorescence staining and Western blotting.Indirect immunofluorescence test showed that the PAb of1:400dilutions still has a higher activity. The PA had no reaction with infectious bovine rhinotracheitis virus, bovine parainfluenza virus type3and bovine adeno virus type3.The PAb can neutralize BVDV and be used in virus neutralization test for BVDVTo study the in vivo replication of BVDV, BALB/c mice were intranasally inoculated with BVDV BA strain. Virus isolation, RT-PCR, immunofluorescence staining,immunohistochemistry(IHC) tests showed that the BVDV strain BA was pathogenic to5week old BALB/c mice. Virus replication in BALB/c mice had occurred in the blood as early as24hours after intranasal inoculation, then the virus could be detected in the lungs of virus-inoculated BALB/c mice. The histopathological examination revealed that alveoli septa thickening was observed in the lungs of virus-inoculated BALB/c mice.In summary, the EO protein of BVDV was expressed successfully and4hybridomas secreting MAbs against EO were obtained immunizing BALB/c mice with the recombinant EO protein.The PAb against EO can neutralize BVDVThe preparation of MAbs and PAb against EOof BVDV lays the basis of establishment of sandwich ELISA for detection of EO.On the other hand. BALB/c mice were intranasally inoculated with BVDV strain BA.The results of pathogen detection and histopathological examination revealed that BVDV could replication in mice and was pathogenic to mice.This experiment lays the basis for the establishment of laboratory animal infection model of BVDV. |
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