[1]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):929-934.[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):929-934.[doi:10.3969/j.issn.1000-4440.2015.04.035]
点击复制

Cry2Aa毒素结合十二肽的筛选与鉴定()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年04期
页码:
929-934
栏目:
加工贮藏·质量安全
出版日期:
2015-08-31

文章信息/Info

Title:
Screening and identification of Cry2Aa-binding peptides from a phage 12-mer peptide library
作者:
武爱华12; 张霄1; 刘媛12; 赵扬1; 赵岩岩12; 张存政1; 谢雅晶1; 刘贤金12
（1.江苏省农业科学院食品质量安全与检测研究所/农业部食品质量安全监控重点开放实验室，江苏南京210014；2.南京农业大学植物保护学院,江苏南京210095)
Author(s):
WU Ai-hua12; ZHANG Xiao1; LIU Yuan12; ZHAO Yang1; ZHAO Yan-yan12; ZHANG Cun-zheng1; XIE Ya-jing1; LIU Xian-jin12
(1.Institute of Food Quality Safety and Detection，Jiangsu Academy of Agricultural Sciences/Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality，Ministry of Agriculture，Nanjing 210014，China;2.College of Plant Protection，Nanjing Agricultural University，Nanjing 210095， China)
关键词:
Cry2Aa毒素; 噬菌体展示肽库; ELISA 
Keywords:
Cry2Aa toxin; phage-display peptide library; ELISA
分类号:
Q785
DOI:
10.3969/j.issn.1000-4440.2015.04.035
文献标志码:
A
摘要:
利用Cry2Aa毒素蛋白对噬菌体展示随机十二肽库进行4轮筛选，并从第4轮筛选产物中随机挑选20个单克隆，进行单克隆 ELISA、PCR扩增、DNA电泳及测序，鉴定这些克隆与 Cry2Aa毒素的结合活性。结果显示，这20个克隆均能与Cry2Aa毒素发生特异性结合，并推导出8条不同的序列。挑取阳性值最高的克隆GT（氨基酸序列为GTPWHHHRHLIV）建立了基于十二肽的Cry2Aa毒蛋白的间接竞争ELISA检测方法。该方法的抑制中浓度（IC50）为1.139 0 μg/ml，最低检测限IC10为0.021 1 μg/ml，线性检测范围为 0.047 8～22.691 0 μg/ml （Y=23.09lgx+48.692，R2=0.996 3）。噬菌体展示肽库筛选方法为快速、准确地检测Cry2Aa毒蛋白提供了新的途径。
Abstract:
A phage displayed random 12-mer peptide library (Ph. D.-12TM Phage Display Peptide Library) is screened with Cry2Aa toxin. After 4 rounds of panning, 20 single colonies were selected randomly,and the positive clones are identified by monoclonal phage competitive enzyme-linked immunosorbent assay (ELISA), and PCR，DNA electrophoresis and sequencing. Totally 8 peptides, which could recognize Cry2Aa specifically have been confirmed. The peptide (amino acid sequence: GTPWHHHRHLIV) holding better binding ability was employed to develop an indirect competitive ELISA for the Cry2Aa detection. The peptide-based indirect competitive ELISA showed that the IC50 was 1.139 0 μg/ml， the minimum detection limit was 0.021 1 μg/ml, and the linear detection ranged from 0.047 8 to 22.691 0 μg/ml. With this new detection method, Cry2Aa can be detected rapidly and accurately．

参考文献/References:


[1]DE MAAGD R A. Bacillus thuringiensis-Based products for insect pest control[M]. Switzerland: Springer International Publishing,2015: 185-192.
[2]DOMINGO J L, GINBORDONABA J. A literature review on the safety assessment of genetically modified plants[J]. Environment International, 2011, 37(4): 734-742.
[3]ANDOW D A, ZWAHLEN C. Assessing environmental risks of transgenic plants[J]. Ecology Letters, 2006, 9(2): 196-214.
[4]LU B R, XIA H. Environmental biosafety of transgenic plants: research and assessment of transgene escape and its potential ecological impacts[J]. Chinese Bulletin of Life Sciences, 2011, 23(2): 186-194.
[5]SANVIDO O, ROMEIS J, GATHMANN A, et al. Evaluating environmental risks of genetically modified crops: ecological harm criteria for regulatory decision-making[J]. Environmental Science & Policy, 2012, 15(1): 82-91.
[6]HEUBERGER S, CROWDER D W, BRVAULT T, et al. Modeling the effects of plant-to-plant gene flow, larval behavior, and refuge size on pest resistance to Bt cotton[J]. Environmental Entomology, 2011, 40(2): 484-495.
[7]ZHANG X, LIU Y, ZHANG C, et al. Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt) Cry1B toxin[J]. Ecotoxicology and Environmental Safety, 2012, 81: 84-90.
[8]WANG Y, ZHANG X, ZHANG C, et al. Isolation of single chain variable fragment (scFv) specific for Cry1C toxin from human single fold scFv libraries[J]. Toxicon, 2012, 60(7): 1290-1297.
[9]华修德,施海燕,王鸣华.噬菌体展示肽库技术及其在农药残留免疫分析中的研究进展[J]. 食品安全质量检测学报,2014,5(12) :3955-3961
[10]LIU R, YU Z, HE Q H, et al. An immunoassay for ochratoxin A with out the mycotoxin[J] . Food Control, 2007, (18): 872-877.
[11]李利红,李常青. 转基因植物中 Bt 杀虫蛋白的重组噬菌体辅助检测[J]. 生物化学与生物物理进展, 2001, 28(5): 728-731.
[12]齐佳. 利用噬菌体展示技术筛选 Cry2Ab 毒素受体结合表位的研究[D]. 北京：中国农业科学院, 2011.
[13]LI Y, WANG Y, ROMEIS J, et al. Bt rice expressing Cry2Aa does not cause direct detrimental effects on larvae of Chrysoperla sinica[J]. Ecotoxicology, 2013, 22(9): 1413-1421.
[14]WANG L H, DENG L H, LAI F X, et al. Optimization of the Cry2Aa Gene and Development of Insect-resistant and Herbicide-tolerant Photoperiod-sensitive Genic Male Sterile Rice[J]. Czech J Genet Plant Breed, 2014, 50(1): 19-25.
[15]MORSE R J, YAMAMOTO T, STROUD R M. Structure of Cry2Aa suggests an unexpected receptor binding epitope[J]. Structure, 2001, 9(5): 409-417.
[16]LENIN K, MARIAM M A, UDAYASURIYAN V. Expression of a cry2Aa gene in an acrystalliferous Bacillus thuringiensis strain and toxicity of Cry2Aa against Helicoverpa armigera[J]. World Journal of Microbiology and Biotechnology, 2001, 17(3): 273-278.
[17]ENGLISH L, ROBBINS H L, VON TERSCH M A, et al. Mode of action of CryIIA: a Bacillus thuringiensis delta-endotoxin[J]. Insect Biochemistry and Molecular Biology, 1994, 24(10): 1025-1035.
[18]徐重新. 人源化抗苏云金芽胞杆菌 Bt (CrylB) 毒素蛋白单链抗体的筛选, 表达及生物学活性测定[D]. 南京：南京师范大学, 2013.
[19]ELLINGTON A D, SZOSTAK J W. Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures[J]. Nature, 1992, 355(6363): 850-852.
[20]DE BRUIN R, SPELT K, MOL J, et al. Selection of high-affinity phage antibodies from phage display libraries[J]. Nature Biotechnology, 1999, 17(4): 397-399.
[21]MOGHADDAM A, LBERSLI I, GEBHARDT K, et al. Selection and characterisation of recombinant single-chain antibodies to the hapten Aflatoxin-B1 from naive recombinant antibody libraries[J]. Journal of Immunological Methods, 2001, 254(1): 169-181.
[22]张青玲,李云河,华红霞,等. Bt 水稻田重要非靶标节肢动物暴露于 Cry2Aa 蛋白的程度分析[J]. Chinese Journal of Applied Ecology, 2013, 24(6): 1647-1651.

相似文献/References:

[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.[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.[doi:doi:10.3969/j.issn.1000-4440.2017.04.034]

备注/Memo

备注/Memo:
收稿日期：2015-01-28 基金项目：国家“973”项目（2012CB722505）；国家自然科学基金项目（3111343、31201535） 作者简介：武爱华（1988-），女，山西忻州人，硕士研究生，从事农药毒理与毒副作用研究。(Tel)18761865017；（E-mail）zhizhuodelei@163.com 通讯作者：刘贤金，(Tel)13805178778;（E-mail）jaasliu@jaas.ac.cn
更新日期/Last Update: 2015-08-31