Prokaryotic Expression of Canine Distemper Virus F Protein and Preparation of Monoclonal Antibody
In order to prepare monoclonal antibody(mAb)against canine distemper virus F protein,the extracellular region of CDV F protein was amplified by RT-PCR,cloned into pET-28a prokaryotic expression vector,and pET-28a-CDV-F recombinant plasmid was constructed.The recombinant plasmid was expressed in BL21(DE3)and induced by IPTG at a final concentration of 0.2 mmol·L-1 at 37℃for 6 h.The expression product was detected by SDS-PAGE.The results showed that the target band appeared at about 33 kDa,and the protein was mainly expressed in the form of inclusion bodies.Western blot results showed that the recombinant F protein could specifically recognize anti-His tag monoclonal antibody and CDV positive serum.The recombinant F protein was purified by gel cutting purification method and used it as immunogen to immunize BALB/c mice for 4 times.The spleen cells were fused with SP2/0 cells.The recombinant F protein was used as the coating antigen to screen the positive hybridoma cells by indirect ELISA and limited dilution method.Finally,two monoclonal antibodies(named 1B6 and 1G8)were screened.The two monoclonal antibodies were identified by antibody subclass kit,and the results showed that the two monoclonal antibodies belonged to IgM,κ type;Western blot analysis of the specificity of two monoclonal antibodies showed that the two monoclonal antibodies specifically recognized recombinant CDV F protein.Both mAbs could react specifically with CDV3 vaccine strain and LN(10)1 wild-type strain by IFA assay.The preparation of monoclonal antibodies against CDV F protein lays a foundation for the detection of CDV.
万方数据
维普
