Wang Hong, Liu Xixia, He Yongsheng, Dong Jiexian, Sun Yuanming, Liang Yan, Yang Jinyi, Lei Hongtao, Shen Yudong, Xu Xiaoyan
Key Laboratory of Food Quality and Safety of Guangdong Province/College of Food Science, South China Agricultural University, Guangzhou 510642, PR China.
Protein Expr Purif. 2010 Jul;72(1):26-31. doi: 10.1016/j.pep.2010.03.001. Epub 2010 Mar 4.
Recombinant antibodies with desirable characteristics that can replace polyclonal or monoclonal antibodies are important for enzyme-linked immunosorbent assay (ELISA) of residues of clenbuterol (CBL), an illicit veterinary drug. Here, we report our work on expression and purification of a mouse-derived anti-CBL single chain Fv (scFv) antibody in Escherichia coli (E. coli). An expression plasmid pBV220-CBL was constructed and transformed into E. coli BL21 (DH3) strain cells. After induction by temperature, the 6x His-tagged anti-CBL scFv antibodies were expressed with the yield of 31%. The solubilized inclusion bodies were extracted, denatured and then purified by Ni-NTA column chromatography. The purified recombinant target protein was analyzed by high performance liquid chromatography, SDS-PAGE and Western blotting, respectively. The results showed the prepared anti-CBL scFv antibodies posed HRP-anti-His-tag antibody-recognized activity and their purity was up to 96%. Moreover, an indirect competitive ELISA based on the anti-CBL scFv antibodies revealed that the limit of detection for CBL was 0.5 ng/ml and the linear range was 1.5-10.6 ng/ml. Taken together, these findings suggest that the prepared recombinant antibody can be used for future immunoassay detection for CBL.
具有理想特性、可替代多克隆或单克隆抗体的重组抗体，对于非法兽药克伦特罗（CBL）残留的酶联免疫吸附测定（ELISA）而言至关重要。在此，我们报告了在大肠杆菌（E. coli）中表达和纯化小鼠源抗CBL单链Fv（scFv）抗体的工作。构建了表达质粒pBV220 - CBL，并将其转化到大肠杆菌BL21（DH3）菌株细胞中。经温度诱导后，表达出了带有6x His标签的抗CBL scFv抗体，产量为31%。提取、变性溶解的包涵体，然后通过Ni - NTA柱层析进行纯化。分别用高效液相色谱、SDS - PAGE和蛋白质免疫印迹法对纯化后的重组目标蛋白进行分析。结果表明，所制备的抗CBL scFv抗体具有HRP - 抗His标签抗体识别的活性，其纯度高达96%。此外，基于抗CBL scFv抗体的间接竞争ELISA显示，CBL的检测限为0.5 ng/ml，线性范围为1.5 - 10.6 ng/ml。综上所述，这些发现表明所制备的重组抗体可用于未来CBL的免疫分析检测。
