Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library

被引：19

作者：

Xu, Chongxin ^{[1
]}

Liu, Xiaoqin ^{[2
]}

Zhang, Cunzheng ^{[1
]}

Zhang, Xiao ^{[1
]}

Zhong, Jianfeng ^{[1
]}

Liu, Yuan ^{[1
]}

Hu, Xiaodan ^{[1
]}

Lin, Manman ^{[1
]}

Liu, Xianjin ^{[1
]}

机构：

[1] Jiangsu Acad Agr Sci, Inst Food Qual Safety & Detect, Key Lab Food Qual & Safety Jiangsu, Prov State Key Lab Breeding Base, Nanjing 210014, Jiangsu, Peoples R China

[2] Huaihua Vocat & Tech Coll, Huaihua 418007, Peoples R China

来源：

ANALYTICAL BIOCHEMISTRY | 2017年 / 518卷

基金：

中国国家自然科学基金;

关键词：

Cry1Ie toxin; Phage display library; Nanobody; Time-resolved fluoroimmunoassay (TRFIA); ANTIBODY FRAGMENT; AFFINITY MATURATION; INSECT RESISTANCE; COTTON BOLLWORM; SELECTION; STRATEGIES; GENE; CORN;

D O I：

10.1016/j.ab.2016.11.006

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08-6.44 ng mL(-1) and the medium inhibition of control (IC50) was 0.73 ng mL(-1). It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%-96.6% and with a coefficient of variation (CV) among 2.0%-8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples. (C) 2016 Elsevier Inc. All rights reserved.

引用

页码：53 / 59

页数：7

共 32 条

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