a College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070 Hubei, People's Republic of China.
Can J Microbiol. 2014 Nov;60(11):737-43. doi: 10.1139/cjm-2014-0468. Epub 2014 Sep 18.
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).
葡萄球菌肠毒素（SEs）是金黄色葡萄球菌合成的强效胃肠道外毒素，可导致多种疾病，包括败血症、食物中毒和中毒性休克综合征以及牛乳腺炎。其中，葡萄球菌肠毒素 A（SEA）是葡萄球菌食物中毒病例中最常见的血清型之一。本研究利用平衡解离常数（KD）为 1.48×10(-8)mol·L(-1)的抗-SEA 单克隆抗体稳定杂交瘤 3C12 建立，获得其 ScFv 编码基因，并在大肠杆菌中表达抗-SEA 单链可变片段（ScFv）蛋白。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、Western blot 和酶联免疫吸附试验对表达的目的 ScFv 蛋白进行了鉴定。结果表明，重组抗-SEA ScFv 蛋白保留了与 SEA 的特异性结合活性，可溶性 ScFv 的 KD 值约为 3.75×10(-7)mol·L(-1)。大肠杆菌瓶培养中生物活性抗-SEA ScFv 的总产率超过 10mg·L(-1)。
