[1]徐重新,张存政,何鑫,等.抗Cry1F毒素单链抗体的筛选及初步应用[J].江苏农业学报,2015,(05):1166-1172.[doi:doi:10.3969/j.issn.1000-4440.2015.05.035]
　XU Chong-xin,ZHANG Cun-zheng,HE Xin,et al.Screening and preliminary application of single-chain fragment variable (scFv) antibody against Cry1F toxin[J].,2015,(05):1166-1172.[doi:doi:10.3969/j.issn.1000-4440.2015.05.035]
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抗Cry1F毒素单链抗体的筛选及初步应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年05期
页码:
1166-1172
栏目:
加工贮藏·质量安全
出版日期:
2015-10-31

文章信息/Info

Title:
Screening and preliminary application of single-chain fragment variable (scFv) antibody against Cry1F toxin
作者:
徐重新; 张存政; 何鑫; 张留娟; 张霄; 仲建锋; 刘媛; 谢雅晶; 刘贤金
(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/农业部农产品质量安全控制技术与标准重点实验室，江苏南京210014)
Author(s):
XU Chong-xin; ZHANG Cun-zheng; HE Xin; ZHANG Liu-juan; ZHANG Xiao; ZHONG Jian-feng; LIU Yuan; XIE Ya-jing; LIU Xian-jin
(Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences/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， Nanjing 210014,China)
关键词:
苏云金芽孢杆菌; Cry1F毒素; 单链抗体; 酶联免疫分析
Keywords:
Bacillus thuringiensis; Cry1F toxin; single-chain fragment variable (scFv) antibody; enzyme-linked immunosorbent assay ( ELISA)
分类号:
Q939.124
DOI:
doi:10.3969/j.issn.1000-4440.2015.05.035
文献标志码:
A
摘要:
对扩增的Tomlinson I 噬菌体抗体库进行3 轮特异性富集，挑取单菌落，采用ELISA、PCR 及测序比对分析鉴定阳性噬菌体单链抗体(scFv)；以宿主替换的方式将阳性噬菌体scFv 基因导入Escherichia coli HB2151 中进行可溶性表达，scFv 过柱纯化后，建立针对Cry1F毒素的IC-ELISA 检测方法。以Cry1B、Cry1C、Cry1Ab、Cry1Ac 作为竞争抑制物，分析scFv的特异性，以Cry1F毒素在玉米中的添加回收试验评价检测方法的实用性。结果显示，从第3 轮富集库中筛选到了11 株具有Cry1F毒素结合活性的噬菌体scFv菌株，经PCR 鉴定都含有目的条带，对其中2株（H1、G9）进行测序，证实为人源化的scFv。选取G9号阳性噬菌体scFv进行可溶性表达，纯化后收集到的scFv浓度为256 μg/ml。建立的IC-ELISA 检测方法检测灵敏度（IC10）为5.99 ng/ml，抑制中浓度（IC50）为0.410 μg/ml，线性检测范围(IC20~IC80)为 0.107~0.713 μg/ml。scFv（G9）对Cry1B、Cry1C具有一定结合活性，交叉反应率分别达到了20.92% 和 15.59%，但不识别Cry1Ab 和Cry1Ac，交叉反应率均低于0.1%。添加回收试验结果表明，基于scFv（G9）建立的IC-ELISA 检测方法稳定性和重复性都比较好。
Abstract:
A large phage antibody library (Tomlinson I) was employed to generate single-chain fragment variable(scFv) antibody against Cry1F toxin by affinity panning, and single colonies were randomly selected from amplified Tomlinson I after 3 rounds of panning. The positive clones were confirmed by ELISA, PCR, and sequencing. The positive phage scFv gene was introduced into Escherichia coli HB2151 by replacement of host for soluble expression. After elution and purification, protein scFv was achieved. An indirect competitive ELISA(IC-ELISA) was then developed for the determination of Cry1F toxin using scFv as antibody. Totally eleven positive clones with distinct nucleotide sequences and intact scFv gene were confirmed to be specific for the Cry1F toxin, among which, H1 and G9 positive clones were the humanized scFv, which were confirmed by DNA sequencing. G9 positive phage was chosen for soluble expression and the concentration of purified scFv reached 256 μg/ml. The concentration of Cry1F toxin spiked in corn sample (IC20~IC80) detected by IC-ELISA ranged from 0.107 to 0.713 μg/ml, and the median inhibitory concentration (IC50 )and detection limit (IC10) were 0.410 μg/ml and 5.99 ng/ml, respectively. The cross-reactivities for Cry1B and Cry1C were 20.92% and 15.59%, and were less than 0.1% for Cry1Ab、Cry1Ac. The results suggested that the IC-ELISA developed in this study had a good repeatability and stability.

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备注/Memo

备注/Memo:
收稿日期：2015-04-07 基金项目：国家自然科学基金项目(31371778)；农业推广项目[TG(14)016] 作者简介：徐重新（1987-），男，湖南永州人，硕士，研究实习员，主要从事食品质量安全与控制技术研究。（E-mail）hhxyxcx@163.com 通讯作者：刘贤金，（E-mail）jaasliu@jaas.ac.cn
更新日期/Last Update: 2015-10-31