抗FLAG标签抗体在植物中的高效表达

High-level expression of anti FLAG tag antibody in plants

摘要:

植物生物反应器是一种新兴的重组蛋白表达系统，是分子农业的核心内容之一。本研究在本氏烟草(Nicotiana benthamiana)中表达了抗八肽(DYKDDDDK, FLAG)标签抗体，并对其进行纯化与鉴定。通过多次免疫小鼠获得高效价抗FLAG抗体并测出其编码序列，然后亚克隆至植物DNA病毒表达载体，最后通过农杆菌介导转染烟草叶片。经Western blotting检测了转染后2−9 d抗体的表达情况：3 d后FLAG抗体开始在烟草叶片中表达，5 d后表达量达到峰值，每千克鲜叶估计可表达66 mg FLAG抗体。抗体经过分离纯化后浓缩为1 mg/mL，按1:10 000稀释仍可识别1 ng/mL的抗原，表明植物生产的FLAG抗体具有高亲和力。植物生物反应器可用于生产高亲和力抗体，并具有简易、成本低和生产周期短等特点，具有很高的应用价值。

Abstract:

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.