Preparation and identification of monoclonal antibodies against non-structural protein C of Peste des petits ruminants virus
To prepare monoclonal antibody against non-structural protein C of Peste des petits ruminants virus(PPRV),a 20-amino acids antigen peptide of CRSGKPRGETPGPLLPEIMQ and a 21-amino acid screening antigen peptide of PLRAGERGLAPQAVQHRTLIK were designed and synthesized based on the antigenicity analysis of the a-mino acid sequence of the polypeptide chain encoded by PPRV C gene.The immunogen and screening antigen were prepared by crosslinking with keyhole limpet hemocyanin(KLH)and biotin carboxyl carrier protein(Biotin),re-spectively.Five female specific pathogen free(SPF)grade BALB/c mice aged 8 to 12 weeks and weighing about 20 g were given intramuscular injection of immunogen for the first immunization,while the second immunization and the third immunization were given at intervals of 7 d after the first immunization,the rush immunization was given at 21 d after the third immunization,and the blood was collected and serum was separated on the third day after the rush immunization.The titer of serum antibody of one immunized mouse was 1∶312 500 and the two mice was 1∶62 500 by indirect enzyme linked immunosorbent assay(ELISA)detection.Three mouse spleen cells were fused with mye-loma cells SP2/0 by polyethylene glycol(PEG)to produce hybridoma cells.26 positive lymphohybridoma cells were screened by indirect ELISA,and 46 monoclonal cell lines were screened by clonal culture.Western blot(WB)screened two hybridoma cell lines that could steadily secrete monoclonal antibodies against PPRV C protein.WB and indirect immunofluorescence assay(IFA)showed that the monoclonal antibodies were of good sensitivity and viral re-activity.These results laid a foundation for further elucidating the role of C protein in the life cycle of PPRV,and al-so provided an effective diagnostic reagent for the detection of peste des petits ruminants(PPR).
Peste des petits ruminants virusnon-structural protein Chybridoma cell fusion techniquemonoclonal antibody
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