[1]武爱华,王耘,刘媛,等.磁珠筛选抗Cry2Aa人源化单链抗体及检测方法的建立[J].江苏农业学报,2017,(04):945-950.[doi:doi:10.3969/j.issn.1000-4440.2017.04.034]
　WU Ai-hua,WANG Yun,LIU Yuan,et al.Screening and detection of Cry2Aa-binding specific single chain antibody fragments(scFv) from a humanized phage display library by magnetic beads[J].,2017,(04):945-950.[doi:doi:10.3969/j.issn.1000-4440.2017.04.034]
点击复制

磁珠筛选抗Cry2Aa人源化单链抗体及检测方法的建立()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年04期
页码:
945-950
栏目:
加工贮藏·质量安全
出版日期:
2017-08-30

文章信息/Info

Title:
Screening and detection of Cry2Aa-binding specific single chain antibody fragments(scFv) from a humanized phage display library by magnetic beads
作者:
武爱华1; 王耘2; 刘媛1; 胡晓丹1; 刘贤金1
（1.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；2.金陵科技学院园艺学院，江苏南京210038)
Author(s):
WU Ai-hua1; WANG Yun2; LIU Yuan1; HU Xiao-dan1; LIU Xian-jin1
(1.Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Food Quality and 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;2.College of Horticulture, Jinling Institute of Technology, Nanjing 210038, China)
关键词:
Cry2Aa毒素; 单链抗体; 磁珠; 间接竞争ELISA
Keywords:
Cry2Aa toxin; specific single chain antibody fragments(scFv); magnetic bead; indirect-competitive ELISA
分类号:
X836
DOI:
doi:10.3969/j.issn.1000-4440.2017.04.034
文献标志码:
A
摘要:
为了建立转基因成分的快速检测方法，利用亲和磁珠偶联 Cry2Aa 毒素蛋白从人源化噬菌体抗体库 Tomlinson I 中液相淘选特异性单链抗体 （scFv）。通过 4 轮“扩增-吸附-洗脱”，从洗脱产物计数板上随机挑取单克隆进行初步鉴定。选取相对结合活性最好的阳性克隆建立间接竞争ELISA，确定其检测范围。筛选后第 4 轮相对产出率较第 1 轮提高了 4.07×106倍；单克隆ELISA鉴定出8个含有完整外源基因片段的阳性克隆，测序后获得3个不同序列的阳性克隆。D5克隆的噬菌体上清对Cry2Aa的检测灵敏度（IC10）为6632 ng/ml，线性范围为 0.016～2881 μg/ml。该克隆对Cry2Aa具有良好的特异性和板内板间重现性。
Abstract:
To develop a rapid detection method of transgenic component in agricultural products, specific single chain antibody fragments (scFv) against Cry2Aa toxin were isolated from humanized phage antibody library (Tomlinson I), by optimizing the magnetic beads and liquid spanning strategy. After 4 rounds of “amplification-adsorption-elution”, single colonies were randomly selected from counting plate of the last round for identification. After DNA sequencing, the positive clones with better binding activity were selected to develop an indirect competitive ELISA, and the detection range of the ELISA was determined. The relative yield of the fourth round of screening was 4.07×106 times higher than that of the first round. Totally 8 positive clones were identified by indirect competitive ELISA, which recognized Cry2Aa specifically and contained the complete gene fragment. The positive clone D5 showed minimum detection limit of 6632 ng/ml and linear range of detection of approximately 0.016-2881 μg/ml ability than others was employed to develop an indirect for Cry2Aa detection by the ELISA method. This method developed in this study has good specificity for Cry2Aa and reproducibility between the plates.

参考文献/References:

[1]JAMES C. 2015 年全球生物技术/转基因作物商业化发展态势]J].中国生物工程杂志，2016，36(4):1-11.
[2]DEVOS Y, AGUILERA J, DIVEKI Z, et al. EFSA′s scientific activities and achievements on the risk assessment of genetically modified organisms (GMOs) during its first decade of existence: looking back and ahead[J]. Transgenic Research, 2014, 23(1): 1-25.
[3]KAMLE S, ALI S. Genetically modified crops: detection strategies and biosafety issues[J]. Gene, 2013, 522(2): 123-132.
[4]NAVARRO E, SERRANO-HERAS G, CASTAO M J, et al. Real-time PCR detection chemistry[J]. Clinica Chimica Acta, 2015, 439: 231-250.
[5]LU J, JI G Z, LI G, et al. Development of a multiplex event-specific PCR assay for detection of genetically modified rice[J]. Cereal Research Communications, 2016, 44(1): 47-56.
[6]SONG L. Application of digital PCR in analyzing transgenic soybeans[C]//Plant and Animal Genome XXIII Conference Plant and Animal Genome America: San Diego, 2015.
[7]SHEN P, GENG F, YU Y, et al. A rapid loop-mediated isothermal amplification method for detection of the modified GM cry1A gene in transgenic insect-resistant cotton and rice[J]. Food Control, 2016, 62: 357-364.
[8]ZHANG X, XU C, ZHANG C, et al. Established a new double antibodies sandwich enzyme-linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a naive mouse phage displayed library[J]. Toxicon, 2014, 81: 13-22.
[9]SANTOS V O, PELEGRINI P B, MULINARI F, et al. A novel immunochromatographic strip test for rapid detection of Cry1Ac and Cry8Ka5 proteins in genetically modified crops[J]. Analytical Methods, 2015, 7(21): 9331-9339.
[10]KUSANO T, OKAMOTO K, MATSUMURA T, et al. Genetically modified mouse expressing a xanthine oxidase mutant that produces a higher ratio of superoxide[J]. Flavins and Flavoproteins, 2011, 2013: 245.
[11]李冬阳,樊凯,吴坚,等. 基于自动磁珠转运的转基因蛋白 Cry1Ab 检测[J]. 分析化学, 2011(9): 5.
[12]LI J, XU Q, WEI X, et al. Electrogenerated chemiluminescence immunosensor for Bacillus thuringiensis Cry1Ac based on Fe3O4@ Au nanoparticles[J]. Journal of Agricultural and Food Chemistry, 2013, 61(7): 1435-1440.
[13]KAMLE S, OJHA A, KUMAR A. Development of enzyme-linked immunosorbent assay for the detection of Bt protein in transgenic cotton[M]// Zhang B H. Transgenic Cotton. New York:Humana Press, 2013:118-125.
[14]CRUZ S, CUBILLOS-ZAPATA C, LPEZ-CALLEJA I M, et al. Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products[J]. Food Control, 2015, 54:322-330.
[15]HAMMERS C M, STANLEY J R. Antibody phage display: technique and applications[J]. Journal of Investigative Dermatology, 2014, 134(2): 17.
[16]SIDHU S S. Phage display in biotechnology and drug discovery[M] Taylor & Francis:CRC Press, 2005.
[17]刘媛,HUOVINEN T,刘贤金,等. 基于磁珠和时间分辨荧光免疫分析的微囊藻毒素LR单链抗体筛选与鉴定[J].中国农业科学, 2011, 45(2): 330-337.
[18]杜方,黄新,纪淑娟. Cry2A蛋白的石英晶体微天平检测[J]. 食品科学, 2014, 35(24): 209-212.
[19]蔡淼,黄新,岳喜庆. 表面等离子共振传感检测Cry2A[J]. 食品科学, 2014, 35(24): 201-204.

相似文献/References:

[1]徐重新,张霄,张存政,等.鼠源噬菌体抗体展示库的构建及初步应用[J].江苏农业学报,2017,(01):210.[doi:10.3969/j.issn.1000-4440.2017.01.034 ]
　XU Chong-xin,ZHANG Xiao,ZHANG Cun-zheng,et al.Construction and preliminary application study of phage display antibody library from mouse[J].,2017,(04):210.[doi:10.3969/j.issn.1000-4440.2017.01.034 ]
[2]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.[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,(04):1166.[doi:doi:10.3969/j.issn.1000-4440.2015.05.035]
[3]刘金凤,吴健敏,覃绍敏,等.抗猪瘟病毒单链抗体基因的构建及其推导的蛋白质三级结构的分子模拟[J].江苏农业学报,2015,(04):817.[doi:10.3969/j.issn.1000-4440.2015.04.017]
　LIU Jin-feng,WU Jian-min,QIN Shao-min,et al.Construction of single chain antibody gene of anti-CSFV and homology modeling of 3D structure of deduced protein[J].,2015,(04):817.[doi:10.3969/j.issn.1000-4440.2015.04.017]
[4]武爱华,张霄,刘媛,等.Cry2Aa毒素结合十二肽的筛选与鉴定[J].江苏农业学报,2015,(04):929.[doi:10.3969/j.issn.1000-4440.2015.04.035]
　WU Ai-hua,ZHANG Xiao,LIU Yuan,et al.Screening and identification of Cry2Aa-binding peptides from a phage 12-mer peptide library[J].,2015,(04):929.[doi:10.3969/j.issn.1000-4440.2015.04.035]
[5]刘媛,刘敏,张霄,等.Cry2A毒素抗独特型单链抗体库的构建[J].江苏农业学报,2018,(05):1174.[doi:doi:10.3969/j.issn.1000-4440.2018.05.029]
　LIU Yuan,LIU Min,ZHANG Xiao,et al.Construction of the anti-idiotypic single-chain variable fragment (scFv) library of Cry2A toxin[J].,2018,(04):1174.[doi:doi:10.3969/j.issn.1000-4440.2018.05.029]

备注/Memo

备注/Memo:
收稿日期：2016-07-12 基金项目：国家自然科学基金项目（3111343、31201535）；江苏省自然科学基金青年基金项目（BK20150114）；江苏省高校自然科学研究项目（14KJB210004）；金陵科技学院博士启动项目（jit-6-201518) 作者简介：武爱华（1988-），女，山西忻州人，硕士，主要从事转基因检测。（E-mail）zhizhuodelei@163.com 通讯作者：刘贤金，（E-mail）jaasliu@jaas.ac.cn
更新日期/Last Update: 2017-09-01