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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2016, Vol. 36 Issue (7): 21-26    DOI: 10.13523/j.cb.20160704
    
Screening of Anti-aflatoxin B1 Single Domain Heavy Chain Antibody Based on Random Mutation of CDR2 and CDR3 Regions
PANG Qian1, MA Yu1, LI Cheng1, LIU Yun-tao1, LIU Shu-liang1, WANG Xiao-hong2, LIU Ai-ping
1 College of Food Science, Sichuan Agricultural University, Yaan 625014, China;
2 College of Food Science and technology, Huazhong Agricultural University, Wuhan 430070, China
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Abstract  

Immunoassay, based on antigen-antibody reaction, was one of the common methods employed to detect AFB1. In order to prepare antibody against AFB1, A codon-optimized DNA sequence (suitable for expression in Escherichia coli (E. coli)) of high homology was synthesized according to comprehensive screening of a large number of anti-AFB1 VHH sequences obtained through phage display library. A phage antibody library was constructed by introducing random mutations in part of CDR2 and CDR3 regions of the synthesized anti-AFB1 VHH. Anti-AFB1 VHH of high affinity was screened through phage-ELISA, using AFB1-OVA as coating antigen. After four rounds of screening, 15 clones capable of binding to AFB1 were obtained. VHH gene of the clone with highest binding ability was amplified, and applied to construct expression plasmid pET-22b-VHH. The corresponding VHH protein was expressed in E. coli BL21(DE3). According to indirect competitive ELISA, the sensitivity of anti-AFB1 VHH obtained was about 10μg/ml.

Key words： VHH      Phage-ELISA      AFB1      Random mutation     
Received: 29 January 2016      Published: 25 July 2016
ZTFLH:  Q511  
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Cite this article:

PANG Qian, MA Yu, LI Cheng, LIU Yun-tao, LIU Shu-liang, WANG Xiao-hong, LIU Ai-ping. Screening of Anti-aflatoxin B1 Single Domain Heavy Chain Antibody Based on Random Mutation of CDR2 and CDR3 Regions. China Biotechnology, 2016, 36(7): 21-26.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20160704     OR     https://manu60.magtech.com.cn/biotech/Y2016/V36/I7/21

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