关键词: 呋喃唑酮；3-氨基-2-噁唑烷酮；单链抗体；结构预测和活性鉴定

Abstract: The gene of single-chain variable fragment (scFv) against 3-amino-2-oxazolidinone (AOZ) derivatives was constructed and physicochemical properties and structure of its encoded protein were predicted in this study. First, total RNA was extracted from hybridoma cell line 1D2 that can secrete a monoclonal antibody (mAb) with high activity and specificity against AOZ derivatives and the heavy and light chain variable region encoding genes (VH and VL) were amplified and connected with each other via a (G4S)3 peptide linker by gene splicing overlap extension-polymerase chain reaction (SOE-PCR) to construct the scFv gene against AOZ derivatives. Then the sequence of amino acids in the antibody was deduced and bioinformatic analysis was carried out after gene sequencing. Finally, the scFv gene was inserted into the expression vector Plip6/GN for expression and the activity of the expressed protein was identified by direct competitive enzyme-linked immunosorbent assay (dcELISA). The results showed that the full length of the scFv gene was 750 bp, encoding a protein of 250 amino acids with a molecular mass of 27 012.20 and a theoretical pI of 9.13. The predicted secondary structure showed that the antibody contained 20 α-helixes, 25 β-turns, 114 random coli and 91 extension bands. The front top view of the tertiary structure prediction showed that the heavy and light chains were connected with each other by linkers to form a circular bucket-like structure, and the side view showed a pocket-like structure, which was consistent with the structural characteristics of natural scFv. The dcELISA results showed that the antibody had good antigen-binding activity. This study will lay a foundation for the establishment of immunoassay methods for AOZ residue detection and the modification of scFv against AOZ derivatives in the future.

Key words: furazolidone; 3-amino-2-oxazolidinone; single chain variable fragment; structure prediction and activity evaluation