[1]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):1174-1182.[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,(05):1174-1182.[doi:doi:10.3969/j.issn.1000-4440.2018.05.029]
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Cry2A毒素抗独特型单链抗体库的构建()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2018年05期
页码:
1174-1182
栏目:
加工贮藏·质量安全
出版日期:
2018-10-25

文章信息/Info

Title:
Construction of the anti-idiotypic single-chain variable fragment (scFv) library of Cry2A toxin
作者:
刘媛1; 刘敏1; 张霄1; 徐重新1; 林曼曼1; 胡晓丹1; 仲建锋1; 谢雅晶1; 罗楚平2; 张存政1; 刘贤金1
（1.江苏省食品质量安全重点实验室，江苏南京210014；2.淮阴工学院生命科学与食品工程系，江苏淮安223003）
Author(s):
LIU Yuan1; LIU Min1; ZHANG Xiao1; XU Chong-xin1; LIN Man-man1; HU Xiao-dan1; ZHONG Jian-feng1; XIE Ya-jing1; LUO Chu-ping2; ZHANG Cun-zheng1; LIU Xian-jin<
(1.Key Laboratory of Food Safety of Jiangsu Province, Nanjing 210014, China;2.Department of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian 223003, China)
关键词:
Cry2A毒素; 抗独特型抗体; 单链抗体; 噬菌体展示
Keywords:
Cry2A toxin; anti-idiotypic antibody; single-chain variable fragment (scFv); phage display
分类号:
X836
DOI:
doi:10.3969/j.issn.1000-4440.2018.05.029
文献标志码:
A
摘要:
为了降低苏云金杆菌Cry2A毒素的免疫检测成本，开发廉价、无毒试剂盒，采用整体抗体免疫和噬菌体展示技术的路线，设计和构建了Cry2A抗独特型单链抗体库。以Protein A纯化的Cry2A兔多克隆抗体为免疫原，对雌性Balb/c系小鼠进行了免疫。在优化的包被原浓度下，用ELISA法测定小鼠血清效价及抗独特型抗体组分。选取效价最高的小鼠，取脾脏提取总RNA，RT-PCR法合成cDNA第一链。设计简并引物，利用PCR法扩增小鼠VH、Vκ基因，再用SOE-PCR法进行单链抗体基因的拼接。SOE-PCR产物和pIT2载体经双酶切后连接，电转入感受态E.Coli. TG1完成建库，并用多克隆噬菌体ELISA对抗体库与免疫原的结合能力进行了鉴定。结果表明，免疫鼠血清的效价在1∶1.6×105到1∶6.4×105倍之间。免疫鼠血清可对Cry2A与其兔多克隆抗体的结合最高可产生17.6%的抑制，证明Ab2β和Ab2γ型抗独特型抗体的存在。最终构建了库容为 1.2×107的Cry2A抗独特型单链抗体库，该抗体库与免疫原的结合信号/背景比值为6.04。
Abstract:
In order to reduce the cost of immunoassay of Bacillus thuringiensis Cry2A toxin and develop a cheap and toxin-free ELISA kit, an anti-idiotypic single-chain variable fragment (scFv) library of Cry2A toxin was designed and constructed by immunization with the whole molecule of rabbit polyclonal antibody and phage display. Rabbit polyclonal antibody against Cry2A toxin was purified by protein A and used as immunogen for the female Balb/c mice. The titer and the component of anti-idiotypic antibodies in mice antiserum were determined by ELISA under the optimized concentration of coating antigens. Then the best mouse was chosen to extract total RNA, and the first strand of cDNA was synthesized by RT-PCR. The VH, Vκ genes of mice and linker fragments were amplified by PCR with designed primers, and the single chain antibody gene was spliced by SOE-PCR. The SOE-PCR product and the pIT2 vector were digested by restriction enzymes and linked together, then were electroporated into competent E.coli.TG1. The binding ability of library with the immunogen was determined by polyclonal phage ELISA. The results showed that the titers of immunized mouse antiserum were between 1∶1.6×105 and 1∶6.4×105. The antiserum could also produce 17.6% inhibition on the binding of Cry2A with its rabbit polyclonal antibodies, and that proved the existence of Ab2β and Ab2γ type anti-idiotypic antibodies. And eventually an anti-idiotypic scFv library was built with the capacity of 1.2×107. The ratio of signal to background of library with immunogen was 6.04.

参考文献/References:

[1]VAN FRANKENHUYZEN K. Insecticidal activity of Bacillus thuringiensis crystal proteins[J]. Journal of Invertebrate Pathology, 2009, 101:1-16.
[2]DONG S, ZHANG C Z, LIU Y, et al. Simultaneous production of monoclonal antibodies against Bacillus thuringiensis (Bt) Cry1 toxins using a mixture immunization [J]. Analytical Biochemistry, 2017, 531:60-66.
[3]YANG Q, TANG S J, RANG J, et al. Detection of toxin proteins from Bacillus thuringiensis strain 4.0718 by strategy of 2D-LC-MS/MS[J]. Current Microbiology, 2015,70:457-463.
[4]NOGUERA P A, IBARRA J E. Detection of new cry genes of Bacillus thuringiensis by use of a novel PCR primer system [J]. Applied and Environmental Microbiology, 2010,76:6150-6155.
[5]GAO H F, WEN L K, WU Y H, et al. An ultrasensitive label-free electrochemiluminescent immunosensor for measuring CrylAb level and genetically modified crops content[J]. Biosensors & Bioelectronics, 2017, 97:122-127.
[6]JIAO L X, LIU Y, ZHANG X, et al. Site-saturation mutagenesis library construction and screening for specific broad-spectrum single-domain antibodies against multiple Cry1 toxins[J]. Applied Microbiology and Biotechnology, 2017, 101:6071-6082.
[7]XU C X, ZHANG X, LIU X Q, et al. Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins[J]. Analytical Biochemistry, 2016, 512:70-77.
[8]HU L, LIU A P, CHEN W F, et al. A non-toxic enzyme-linked immunosorbent assay for aflatoxin B1 using anti-idiotypic antibodies as substitutes [J]. Journal of the Science of Food and Agriculture, 2017, 97:1543-1548.
[9]WANG D, XU Y, TU Z, et al. Isolation and characterization of recombinant variable domain of heavy chain anti-idiotypic antibodies specific to aflatoxin B-1[J]. Biomedical and Environmental Sciences, 2014, 27:118-121.
[10]TSUTSUMI T, NAGATA S, YOSHIDA F, et al. Anti-idiotype monoclonal antibodies against anti-microcystin antibody and their use in enzyme immunoassay[J]. Toxicon, 1998, 36:235-245.
[11]LIU B H, YU F Y, CHU F S. Anti-idiotype and anti-anti-idiotype antibodies generated from polyclonal antibodies against microcystin-LR[J]. Journal of Agricultural and Food Chemistry, 1996, 44:4037-4042.
[12]MARAGOS C M. Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms[J]. Mycotoxin Research, 2014, 30:103-111.
[13]YU F Y, CHU F S. Production and characterization of a monoclonal anti-anti-idiotype antibody against fumonisin B(1) [J]. Journal of Agricultural and Food Chemistry, 1999, 47:4815-4820.
[14]江涛，郑佳，李楠, 等. 抗T-2毒素单抗独特型抗体的制备及应用研究[J]. 中国食品卫生杂志, 2007,19(3): 234-237.
[15]LAN H N, ZHENG X, KHAN M A, et al. Anti-idiotypic antibody: A new strategy for the development of a growth hormone receptor antagonist[J]. International Journal of Biochemistry & Cell Biology, 2015, 68:101-108.
[16]刘媛，梁颖，王耘，等. 抗独特型抗体在小分子农药、真菌毒素免疫检测中的应用[J]. 浙江农业学报，2010, 23 (3): 398-402.
[17]XU Y, XIONG L, LI YP, et al. Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library[J]. Analytical and Bioanalytical Chemistry, 2015, 407:5333-5341.
[18]XU Y, XIONG L, LI Y P, et al. Citrinin detection using phage-displayed anti-idiotypic single-domain antibody for antigen mimicry[J]. Food Chemistry, 2015, 177:97-101.
[19]GOLETZ S, CHRISTENSEN P A, KRISTENSEN P, et al. Selection of large diversities of antiidiotypic antibody fragments by phage display[J]. Journal of Molecular Biology, 2002, 315:1087-1097.
[20]ZHOU H Y, ZHANG Y L, LU G D, et al. Recombinant antibody libraries and selection technologies[J]. New Biotechnology, 2011, 28:448-452.
[21]伯吉斯,多伊彻. 蛋白质纯化指南[M]. 陈薇,译.北京:科学出版社, 2009:383-460.
[22]CLACKSON T, HOOGENBOOM H R, GRIFFITHS A D, et al. Making antibody fragments using phage display libraries[J]. Nature, 1991, 352:624-628.
[23]ORLANDI R, GUSSOW D H, JONES P T, et al. Cloning immunoglobulin variable domains for expression by the polymerase chain-reaction[J]. Proceedings of the National Academy of Sciences of the United States of America, 1989, 86:3833-3837.
[24]PASELLO M, ZAMBONI S, MALLANO A, et al. Design and construction of a new human naive single-chain fragment variable antibody library, IORISS1[J]. Journal of Biotechnology, 2016, 224:1-11.
[25]DE WILDT R M T, MUNDY C R, GORICK B D, et al. Antibody arrays for high-throughput screening of antibody-antigen interactions[J]. Nature Biotechnology, 2000, 18:989-994.
[26]李敏,计融. 抗独特型抗体的研究现状[J]. 中国预防医学杂志, 2006, 7(2):147-149.
[27]蒂姆克拉克森,亨利洛曼. 噬菌体展示: 通用实验指南[M]. 马岚, 卢帅，李铭, 译. 北京:化学工业出版社, 2004:147-168.
[28]徐重新,张霄,张存政,等. 鼠源噬菌体展示库的构建及初步应用[J].江苏农业学报, 2017,33（1）：210-217.
[29]郑志明,金社胜,肖希龙,等. 抗诺氟沙星噬菌体单链抗体库的构建与筛选[J]. 动物医学进展，2010，31(S1):36-41.

备注/Memo

备注/Memo:
收稿日期：2017-11-09 基金项目：国家自然科学基金重点项目（31630061）；江苏省社会发展项目（BE2017706）；江苏省农业科技自主创新基金项目[CX（17）3018]；中央财政农业推广项目[TG(16)-031]；江苏省自然科学基金项目（BK20181247） 作者简介：刘媛（1980-），安徽合肥人，女，硕士，副研究员，主要从事农产品污染物生物控制技术研究。（Tel）025-84390401；（E-mail）zeranol@163.com 通讯作者：刘贤金, （Tel）025-84390401；（E-mail）jaasliu@jaas.ac.cn
更新日期/Last Update: 2018-11-05