鲨鱼单域抗体融合蛋白的克隆表达、稳定性及检测性能研究

Study on the cloning, expression, stability and detection performance of shark-derived single domain antibody fusion proteins

中文关键词:  单域抗体  恩诺沙星  稳定性  酶联免疫吸附方法  水产品

英文关键词:single-domain antibodies  enrofloxacin  stability  enzyme-linked immunosorbent assay  aquatic products

中文摘要:

      目的 探究鲨鱼单域抗体融合蛋白的克隆表达及其性能。方法 通过大肠杆菌表达系统高效表达鲨鱼单域抗体融合蛋白2E6-SUMO; 以传统的免疫球蛋白G (immolunoglobulin G, IgG)抗体为对照, 对其热稳定性及酸碱稳定性进行研究; 并以2E6-SUMO为识别元件, 建立间接竞争酶联免疫吸附法, 并应用于水产品中恩诺沙星残留的检测。结果 2E6-SUMO的可溶性表达量为1.67 mg/L, 与传统的IgG抗体相比, 2E6-SUMO具有更好的热稳定性和酸碱稳定性; 建立了基于2E6-SUMO检测恩诺沙星的间接竞争酶联免疫吸附法, 半抑制浓度(half maximal inhibitory concentration, IC50)为42.08 ng/mL, 检出限为3.84 ng/mL, 线性范围为9.94~376.17 ng/mL; 鱼肉基质对2E6-SUMO没有显著干扰, 加标样品的回收率在83.33%~123.06%之间。结论 鲨鱼单域抗体融合蛋白实现了高效表达, 表现出更好的稳定性, 可作为一种新型特异性免疫元件用于水产品中药物残留的免疫检测。本研究为鲨鱼单域抗体融合蛋白的应用提供了参考。

英文摘要:

      Objective To explore the cloning, expression and performance of shark single domain antibody fusion proteins. Methods Shark-derived single domain antibody fusion protein 2E6-SUMO was efficiently expressed by Escherichia coli expression system; its thermal and acid-base stability was investigated using conventional immolunoglobulin G (IgG) antibody as a control, and constructed an indirect competition enzyme-linked immunosorbent assay using 2E6-SUMO as the recognition element, and applied it to the detection of enrofloxacin residues in aquatic products. Results The soluble expression level of 2E6-SUMO was 1.67 mg/L, and the 2E6-SUMO had better thermal and acid-base stability compared with the traditional IgG antibody, indirect competitive enzyme-linked immunosorbent assay based on 2E6-SUMO for the detection of enrofloxacin was developed with an half maximal inhibitory concentration (IC50) value of 42.08 ng/mL, the limit of detection was 3.84 ng/mL, with a linear range of 9.94?376.17 ng/mL; and the experiments showed that the fish matrix did not significantly interfere with 2E6-SUMO, and the recoveries of spiked samples ranged from 83.33% to 123.06%. Conclusion The shark-derived domain antibody fusion protein achieve efficient expression, shows better stability, and can be used as a novel specific immolune element for immunodetection of drug residues in aquatic products. This study provides a reference for the application of shark-derived single domain antibody fusion protein.