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2025年8月29日 星期五
农业生物技术学报  2019, Vol. 27 Issue (3): 516-525    DOI: 10.3969/j.issn.1674-7968.2019.03.016
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
单链抗体对Cry1A类毒素的检测方法建立及识别差异初步研究
曲婷婷1,2, 张霄2, 董飒2, 刘贝贝2, 李盼2, 王耘2, 张存政2, 刘贤金1,2,*
1 南京农业大学 植物保护学院,南京 210095;
2 江苏省农业科学院 食品质量安全与检测研究所/江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地/农业部农产品量安全控制技术与标准重点实验室,南京 210014
Construction of scFv Antibody for the Determination of Cry1A Toxins and Preliminary Study on Their Interaction Difference
QU Ting-Ting1,2, ZHANG Xiao2, DONG Sa2, LIU Bei-Bei2, LI Pan2, WANG Yun2, ZHANG Cun-Zheng2, LIU Xian-Jin1,2,*
1 College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China;
2 Key Laboratory of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base/Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture / Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
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摘要 为建立一种针对Cry1类毒素的经济高效的检测方法,本研究以杂交瘤细胞株2D10 cDNA为模板,通过重叠延伸PCR(gene splicing by overlap extension PCR, SOE-PCR)技术构建单链抗体(single-chain variable fragment, scFv)基因,转入大肠杆菌(Escherichia coli) BL21 (DE3)表达并建立双抗体夹心酶联免疫吸附测定(double antibody sandwich ELISA, DAS-ELISA)检测方法,通过分子对接模拟分析影响scFv与Cry1A类毒素结合的关键因素。结果成功构建了scFv基因,表达纯化出具有较高识别活性的scFv抗体(约28 kD),所建立的DAS-ELISA方法对Cry1Ab/Cry1Ac毒素的最低检测限(limits of determination, LOD)为20 ng/mL,分子对接结果显示,毒素的三维结构决定了其结合活性,氢键和疏水作用力在scFv与毒素结合过程中起重要作用。本研究利用基因工程抗体技术构建并表达获得了scFv抗体,建立了针对Cry1Ab/Cry1Ac毒素的双抗夹心ELISA检测方法,初步分析研究了scFv与Cry1Ac/Cry1Ab和Cry1Aa的识别差异机制,为进一步改造、研究开发新型广谱scFv提供了理论依据。
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曲婷婷
张霄
董飒
刘贝贝
李盼
王耘
张存政
刘贤金
关键词 Cry1AbCry1AcscFvDAS-ELISA分子对接    
Abstract:To establish a cost-effective detection method for Cry1 toxins, a hybridoma cell line 2D10 was used as a cDNA template to construct a single-chain antibody (scFv) gene via gene splicing by overlap extension PCR (SOE-PCR). After being transferred into Escherichia coli BL21 (DE3), the scFv gene was expressed and a double antibody sandwich ELISA (DAS-ELISA) was established. Meanwhile, the key factors affecting the interaction of scFv to Cry1A toxins were analyzed by molecular docking simulation. As a result, the scFv gene was successfully constructed and the scFv antibody with high activity was purified (about 28 kD). The minimum detection limit (LOD) of the established DAS-ELISA for Cry1Ab/Cry1Ac toxin was 20 ng/mL. The docking results showed that the three-dimensional structure of the toxin determined its binding activity, hydrogen bonds and hydrophobic interaction played an important role during the binding process between scFv and Cry1A toxins. In this study, a scFv was constructed and expressed based on genetic engineering antibody technology, a DAS-ELISA was established for Cry1Ab/Cry1Ac toxin determination and the recognization mechanism difference of scFv to Cry1Ac/Cry1Ab and Cry1Aa was preliminarily analyzed which provide a theoretical basis for the research and development of a new broad-spectrum scFv.
Key wordsCry1Ab    Cry1Ac    scFv    DAS-ELISA    Molecular docking
收稿日期: 2018-08-31     
ZTFLH:  S3  
基金资助:国家自然科学基金项目(No. 31371778和No. 31301703)、江苏省自主创新项目(No. CX(14)5068)和江苏省自然科学基金(No. BK20150114)
通讯作者: *通迅作者,jaasliu@163.com   
引用本文:   
曲婷婷, 张霄, 董飒, 刘贝贝, 李盼, 王耘, 张存政, 刘贤金. 单链抗体对Cry1A类毒素的检测方法建立及识别差异初步研究[J]. 农业生物技术学报, 2019, 27(3): 516-525.
QU Ting-Ting, ZHANG Xiao, DONG Sa, LIU Bei-Bei, LI Pan, WANG Yun, ZHANG Cun-Zheng, LIU Xian-Jin. Construction of scFv Antibody for the Determination of Cry1A Toxins and Preliminary Study on Their Interaction Difference. 农业生物技术学报, 2019, 27(3): 516-525.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.03.016     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I3/516
 
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