title: Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.
abstract: The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10(-8) mol·L(-1), its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10(-7) mol·L(-1). The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L(-1).
authors.affiliation: a College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070 Hubei, People's Republic of China.
authors.forename: Weifeng
authors.initials: W
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Hu
authors.forename: Aiping
authors.initials: A
authors.lastname: Liu
authors.forename: Jinquan
authors.initials: J
authors.lastname: Li
authors.forename: Fusheng
authors.initials: F
authors.lastname: Chen
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D050438:Genes, Immunoglobulin Heavy Chain; D050439:Genes, Immunoglobulin Light Chain; D006825:Hybridomas; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D013211:Staphylococcus aureus / Q000276:immunology*
chemical_list: D004768:Enterotoxins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C026066:enterotoxin A, Staphylococcal
keywords: ELISA; anticorps à chaîne unique du fragment variable; entérotoxine staphylococcique de type A; expression; hybridoma; hybridome; single chain Fv antibody; staphylococcal enterotoxin A