Department of Microbiology and Immunology, Faculty of Medicine and health sciences, Alneelain University, P.O. Box 12702, Khartoum, Sudan.
Biochem Genet. 2012 Aug;50(7-8):625-41. doi: 10.1007/s10528-012-9506-4. Epub 2012 May 3.
Single-chain variable fragment (scFv) is one of the most common antibody forms. This report describes the expression of the scFv gene as a soluble protein in Origami DE3 cytoplasm. The purified scFv recognized the epidermal growth factor receptor (EGFRvIII) on the surface of MCF-7 cells. The scFv protein was purified in soluble form at a concentration of 10 mg/l, and the scFv protein activity and specificity were characterized using several immunological assays. The purified scFv protein showed specific binding to MCF-7 cells, evidenced by a band of 68 kDa in Western blot analysis, and immunofluorescence clearly proved that the scFv antibody recognized the EGFRvIII antigen epitopes. Furthermore, 53 % of the MCF-7 cells were bound to scFv protein, as measured by flow cytometry analysis. This study demonstrated that the Origami DE3 expression system can produce single-chain antibodies in active form for later use in gene therapy and vaccine production.
单链可变片段 (scFv) 是最常见的抗体形式之一。本报告描述了 scFv 基因在 Origami DE3 细胞质中作为可溶性蛋白的表达。纯化的 scFv 识别 MCF-7 细胞表面的表皮生长因子受体 (EGFRvIII)。scFv 蛋白以 10mg/L 的浓度以可溶性形式纯化，并用几种免疫学测定法对 scFv 蛋白的活性和特异性进行了表征。纯化的 scFv 蛋白显示出与 MCF-7 细胞的特异性结合，Western blot 分析显示出 68kDa 的条带，免疫荧光清楚地证明了 scFv 抗体识别 EGFRvIII 抗原表位。此外，通过流式细胞术分析，53%的 MCF-7 细胞与 scFv 蛋白结合。这项研究表明，Origami DE3 表达系统可以产生具有活性的单链抗体，用于以后的基因治疗和疫苗生产。
