Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cryl F Toxin Single-Chain Antibodies
Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cryl F Toxin Single-Chain Antibodies
复制标题
构建免疫兔噬菌体展示文库筛选高活性苏云金芽孢杆菌Cryl F毒素单链抗体
DOI：
10.1021/acs.jafc.7b01985
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发表时间：
2017
影响因子：
6.1
通讯作者：
Liu Xianjin
中科院分区：
文献类型：
--
作者：
Xu Chongxin;Zhang Cunzheng;Zhong Jianfeng;Hu Hui;Luo Shimin;Liu Xiaoqin;Zhang Xiao;Liu Yuan;Liu Xianjin
In the present study, a Cry1F-immunized rabbit phage display library (6.96 × 108cfu/mL) was constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (a single-chain variable fragment, scFv) by biopanning. A total of 16 positive monoclonal phage scFv’s were obtained after 4 rounds of panning, which were identified by enzyme-linked immunosorbent assay (ELISA), polymerized chain reaction, and DNA sequencing. The most positive phage scFv (named RF4) was expressed inEscherichia coliHB2151, and a soluble protein of approximately 30 kDa was purified with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. An indirect competitive ELISA (IC-ELISA) was established on the basis of purified soluble RF4-scFv for Cry1F toxin. It indicated the 50% inhibition of the control (IC50) was 11.56 ng/mL and the detection limit (IC10) was 0.18 ng/mL and showed weak cross-reactivities for Cry1Ab (2.8%), Cry1Ac (1.3%), and Cry1B, Cry1C, Cry1Ie, and Cry2A (less than 0.1%). It was found that IC-ELISA detected Cry1F toxin spiked in rice, wheat, corn, and soil samples with good accuracy, stability, and repeatability. The recoveries were in the range of 80.2–99.6%, and the coefficients of variation were in the range of 2.5–10.0%. These results showed that IC-ELISA based on scFv from the immunized rabbit phage display library was promising for specific detection of Cry1F toxin in agroproducts and environmental samples.
