Screening and analysis of mimic epitopes of monoclonal antibody 4H3 against Brucella canis ZG strain

Abstract:

[background] At present, there are some difficulties in the diagnosis of Brucella canis. [Objective] To screen and analyze the specific epitopes of monoclonal antibody 4H3 against Brucella canis. [Methods] The phage display peptide library was used for screening. The 4H3 against B. canis was used as the target molecule, which was employed to coat the ELISA plate. Then, we applied 12-mer phage display peptide library for screening through 3 rounds of biopanning. In the biopanning, yield of phage increased from 5.00×10^-7 to 9.84×10^-6, and the false positive rate gradually decreased. We selected 14 of the screened positive clones for amplification, followed by extraction and sequencing of the genomic DNA. The affinity and specificity of the positive clones were detected by iELISA and cELISA. [Results] There were 3 different kinds of short peptide sequences in 14 monoclonal phages, which were KMSIRHPIRLPI, ILRRRRKRIIQI, and QRIHMRLTTQS, respectively. The affinity of the 3 short peptide sequences to monoclonal antibodies was in the order of KMSIRHPIRLPI>ILRRRRKRIIQI>QRIHMRLTTQS, and KMSIRHPIRLPI and ILRRRRKRIIQI had strong specificity. Thus, we further analyzed KMSIRHPIRLPI and ILRRRRKRIIQI and found that KMSIRHPIRLPI showed high similarity to peptidase C26 family protein (PuuD) of B. canis RM6/66 (75% similarity in amino acids) with 4 consecutive similar amino acids. ILRRRRKRIIQI had high similarity to 3-oxoyl-[acyl carrier protein] reductase (OAR) of B. canis RM6/66 (75% similarity in amino acids), with 2 consecutive similar amino acids. [Conclusion] Based on phage display peptide library, the short peptide sequences specifically binding to the monoclonal antibody against B. canis ZG were screened out.

Key words:Brucella canis;monoclonal antibody;12-mer phage display peptide library;epitope