Abstract：The objective of this study is to develop and characterize monoclonal antibodies against duck avian β-defensin 9 (AvBD9). Firstly, the pGEX-duck AvBD9 recombinant protein was used to immunize the female BALB/c Mus musculus (6~8 weeks) three times every two weeks followed by boost immunization 3 d before fusion. Then the duck-AvBD9 gene was subcloned into EcoRⅠand SalⅠrestriction sites of prokaryotic pET-32a vector to construct a recombinant plasmid pET-32a-duck AvBD9. The expression of protein in Escheriohia coli BL21 (JM83-) containing the recombinant plasmid was induced by IPTG followed by SDS-PAGE analysis. The molecular weight of the recombinant protein was 26 kD. The pET-32a-duck AvBD9 protein was purified by nickel affinity chromatography. Mouse antiserum positive against pGEX-duck AvBD9 protein could also recognize pET-32a-duck AvBD9 protein. MAb (monoclonal antibody) against duck AvBD9, designated as 1F11, was finally identified by indirect ELISA and Western blotting assays using the recombinant protein pET-32a-duck AvBD9. The MAb 1F11 could specifically recognize pGEX-duck AvBD9, from pGEX-duck AvBD10 protein, pGEX-chicken AvBD10 protein or pGEX-6p-1 protein. The results of isotype analysis showed that MAb 1F11 belonged to IgG1 subclass and the light chain is the κ chain. The MAb 1F11 with good specificity can be used for further research of duck AvBD9 protein.
