[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):210-217.[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,(01):210-217.[doi:10.3969/j.issn.1000-4440.2017.01.034 ]
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鼠源噬菌体抗体展示库的构建及初步应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年01期
页码:
210-217
栏目:
加工贮藏·质量安全
出版日期:
2017-02-28

文章信息/Info

Title:
Construction and preliminary application study of phage display antibody library from mouse
作者:
徐重新; 张霄; 张存政; 刘媛; 仲建锋; 董飒; 胡晓丹; 林曼曼; 刘贤金
(江苏省农业科学院食品质量安全与检测研究所，省部共建国家重点实验室培育基地——江苏省食品质量安全重点实验室，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 Xiao; ZHANG Cun-zheng; LIU Yuan; ZHONG Jian-feng; DONG Sa; HU Xiao-dan; LIN Man-man; LIU Xian-jin
(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, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
关键词:
噬菌体展示库; 单链抗体; 重叠延伸PCR; 噬菌粒载体; Bt毒素
Keywords:
phage display library; single chain antibody（ScFv）; gene splicing by overlapping extension PCR(SOE- PCR); phagemid vector; Bt toxins
分类号:
Q936
DOI:
10.3969/j.issn.1000-4440.2017.01.034
文献标志码:
A
摘要:
构建可用于特异性单链抗体（ScFv）筛选和应用的大容量鼠源噬菌体抗体展示库，获得拥有自主知识产权的抗体库材料。从小鼠脾脏细胞中提取总RNA，反转录成cDNA后，分别扩增抗体的重链基因、轻链基因，并设计2条互补的Linker基因。采用重叠延伸PCR （SOE-PCR）法将重链-Linker和Linker-轻链拼接为完整的ScFv基因，酶切、酶连后，将ScFv基因克隆到pCANTAB-5E噬菌粒载体上，经电穿孔导入E.coli TG1感受态细胞中，过夜培养获得初级噬菌体展示库。以单克隆菌落数计算库容量大小，通过比对随机挑取的单克隆噬菌体ScFv可变区序列的差异，分析库的多样性。以Bt毒素（Cry1B、Cry1C、Cry1F）为包被抗原，从库中筛选具有结合活性的ScFv验证库的实用性。经检测，提取的总RNA条带清晰，浓度为2.76 μg/μl，扩增的抗体重链基因、轻链基因条带清晰，且2条互补的Linker基因成功添加到重链基因和轻链基因上。经SOE-PCR法扩增，重链-Linker与Linker-轻链成功拼接为ScFv基因。电转化后，经PCR（Polymerase chain reaction）扩增和凝胶电泳检测，证实ScFv基因克隆成功，测得初级噬菌体抗体展示库的库容为 3.67×108。随机挑取12个单克隆菌种进行测序和比对，证实ScFv基因为鼠源抗体基因，且12个单克隆菌种的ScFv基因可变区均有一定差异，说明ScFv基因具有多样性。以3种Bt毒素(Cry1B、Cry1C、Cry1F)为抗原，经4轮特异性富集筛选后均获得了具有抗原结合活性的噬菌体ScFv菌种，表明该库的实用性较强。
Abstract:
A large phage display antibody library of mice for ScFvs screening and application research was constructed to obtain the independent intellectual property of the library. Extraction total RNAs from spleen cells of mice, then reverse transcribed them to cDNAs. Amplification of heavy chain gene (VH) and light chain gene (VL) fragments with PCR, design two complementary Linker sequences and add to VH and VL respectively. Splicing VH-Linker and Linker-VL sequences by overlapping extension PCR (SOE-PCR) to a whole ScFv. Then clone ScFv into the pCANTAB-5E phagemid vectors，after being digested by restriction endonuclease ( Not I、Sfi I) and linked by T4 DNA ligase. Obtained primary phage display antibody library with over-night cultured that the pCANTAB-5E-ScFv recombinant plasmid introduced into E.coli TG1 by electroporation. The capacity of this library was calculated by monoclonal colonies. To analyze the diversity of the library, the amino acid sequences of monoclonal colony phage ScFv in variable regions were alignment. We made Bt toxin proteins as coating antigen to verify its practicality by screening ScFvs which had antigen binding activity from the library. After detection, the total RNAs had clear bands and its concentration was 2.76 μg/μl. We could clearly find bands of VH and VL by agarose gel electrophoresis and their complementary Linker genes were added respectively. Finally, the VH-Linker and VL-Linker were spliced to ScFvs by SOE-PCR and cloned into the pCANTAB-5E phagemid vector successfully. We obtained the primary phage display antibody library with a capacity of 3.67×108. By sequencing and sequence alignment of 12 monoclonal colonies phage ScFvs from random selection, we could determin the ScFv were from mice and the library had high diversity. After four rounds of panning in the library with three kinds of Bt(Cry1B、Cry1C、Cry1F), the ScFvs which could combine with Cry1B、Cry1C、Cry1F were obtained. The results showed that this library had a stronger practicality.

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备注/Memo

备注/Memo:
收稿日期：2016-05-06 基金项目：国家自然科学基金项目（31630061、31371778）；省部共建国家重点实验室培育基地——江苏省食品质量安全重点实验室自主研究课题（49114063201604、49114063201608）；江苏省农业科学院院基金项目（6111676） 作者简介：徐重新(1987-)，男，湖南永州人，硕士，助理研究员，从事食品质量安全检测技术研究。(E-mail)hhxyxcx@163.com 通讯作者：刘贤金，（E-mail）jaasliu@jaas.ac.cn
更新日期/Last Update: 2017-04-12