A single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein for glycocholic acid (GCA) was produced and characterized. The scFv gene with a 218 linker was generated by splicing by overlap extension (SOE)-polymerase chain reaction (PCR) and sequentially inserted into the expression vector pecan45 containing AP gene to express the scFv-AP fusion protein in Escherichia coli (E. coli). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the fusion protein showed the expected molecular weight of about 80 kDa. Both the antibody binding capacity and AP enzyme activity of the scFv-AP fusion protein were validated by colorimetric analysis. One-step competitive direct enzyme-linked immunosorbent assay (ELISA) based on the scFv-AP fusion protein indicated that the average concentration required for 50% inhibition of binding (IC₅₀) and limit of detection (LOD) for GCA were 216 ng mL⁻¹ and 37.0 ng mL⁻¹, respectively, and the linear response range extended from 71.0 to 657 ng mL⁻¹. The cross-reactivity (CR) of the scFv-AP fusion protein was similar to those of its parental scFv antibody. The scFv-AP fusion protein was bifunctional, retaining both antibody binding specificity and AP enzyme activity. This work indicates that the production of the scFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggests that it could be further used as convenient one-step detection probes for GCA.

中文翻译：

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单步酶联免疫吸附测定中用于糖胆酸检测的单链可变片段碱性磷酸酶融合蛋白的生产与表征

产生并表征了单糖胆酸（GCA）的单链可变片段（scFv）-碱性磷酸酶（AP）融合蛋白。通过剪接产生通过重叠延伸（SOE） -聚合酶链式反应（PCR），并依次插入到含有AP基因表达在所述scFv-AP融合蛋白的表达载体pecan45与218接头的scFv基因的大肠杆菌（大肠杆菌）。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，融合蛋白显示预期的分子量约为80 kDa。通过比色分析验证了scFv-AP融合蛋白的抗体结合能力和AP酶活性。基于scFv-AP融合蛋白的一步竞争性直接酶联免疫吸附测定（ELISA）表明，抑制50％结合所需的平均浓度（IC ₅₀）和对GCA的检出限（LOD）为216 ng mL ^-1和37.0 ng mL ^-1，线性响应范围从71.0扩展到657 ng mL ^-1。scFv-AP融合蛋白的交叉反应（CR）与其亲本scFv抗体的交叉反应相似。scFv-AP融合蛋白具有双重功能，既保留抗体结合特异性，又保留AP酶活性。这项工作表明在大肠杆菌菌株BL21（DE3）pLysS中生产scFv-AP融合蛋白是可行的，并表明它可以进一步用作GCA的便捷一步检测探针。