牛结节性皮肤病病毒ORF122蛋白单克隆抗体的制备与鉴定OA

This study aimed to prepare and characterize a recombinant ORF122 protein of lumpy skin disease virus(LSDV)and its monoclonal antibodies(mAbs).The extracellular region of the ORF122 pro-tein(AA68-196)was cloned into a prokaryotic expression vector,expressed in Escherichia coli and pu-rified to obtain the recombinant protein rLSDV122.BALB/c mice were immunized with rLSDV122,and hy-bridoma technology was used to screen for ORF122-specific mAb-secreting cell lines.The isotypes and indirect ELISA titers of the obtained mAbs were determined,and their ability to recognize native ORF122 in infected cells as well as potential cross-reactivity with common bovine infectious pathogens was evaluated by Western-blot and indirect immunofluorescence assay.The results showed that a recom-binant protein with a molecular mass of approximately 17 kDa,designated rLSDV122,was successfully ob-tained,together with two IgG1/κ mAbs,2D5 and 4H3,whose endpoint titers were 1∶128 000 and 1∶64 000,respectively.Immunological analyses demonstrated that both mAbs specifically recognized native ORF122 in LSDV-infected MDBK cells and did not cross-react with several common bovine infectious pathogens.These findings indicate that high-specificity mAbs against LSDV ORF122 were successfully produced,providing a foundation for etiological studies of LSDV and for the development of related im-munodiagnostic assays.