抗AFB_1单域重链抗体文库淘选方法的研究

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抗AFB_1单域重链抗体文库淘选方法的研究
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基金项目:食品科学与技术国家重点实验室目标导向资助项目(SKLF-MB-201002);国家中小企业创新基金项目(09C26213604449)

Research on Panning Methods of Single-Domain Heavy-Chain Antibody Fragments for Aflatoxin B1 from Non-Immune Library

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摘要:

比较两种从噬菌体展示文库中淘选抗黄曲霉毒素B1单域重链抗体的方法,并为淘选针对小分子物质的单域重链抗体提供参考。采用固相淘选技术,以黄曲霉毒素B1人工抗原为靶分子,淘选天然单域重链抗体库,分别采用Gly-HCl法和AFB1竞争法洗脱结合噬菌体,对洗脱物进行滴度测定,以回收率和富集度为指标,对两种洗脱方法进行比较。采用phage-ELISA法鉴定噬菌体克隆,并对阳性克隆进行交叉反应分析以及序列测定。滴度测定结果显示,Gly-HCl法回收率比竞争法高5～10倍。分别随机挑取20个单克隆进行phage-ELISA鉴定,其中,Gly-HCl洗脱法未获得阳性克隆,AFB1竞争洗脱法得到8个阳性克隆。序列测定结果显示,这8个克隆均编码单域重链抗体。

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The objective of this study was tocompare the efficiency of two elute strategies in biopanning of single-domain heavy-chain antibody(HCAbs) phage display library against low molecular hapten,Aflatoxin B1(AFB1).For this,the artificial antigen AFB1-BSA and AFB1-OVA were used as ligand to screen the binding phages from an Alpaca non-immune library by antigen-coated solid phase selection.The binding phages were directly eluted with Gly-HCl or competitively eluted with AFB1 solution.The eluants were tittered separately.It was found that the recovery rate of Gly-HCl is 5 to 10 times higher than that of competition method.Twenty colons randomly picked from each eluant were characterized by phage-ELISA.Eight positive colons were isolated from the eluant by the competition method;however,no positive colons were isolated by Gly-HCl method.The sequencing data show that all the colons are encoding single-domain HCAbs.

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刘夏,许杨,涂追,何庆华.抗AFB_1单域重链抗体文库淘选方法的研究[J].食品与生物技术学报,2011,30(6):950-955.

LIU Xia, XU Yang, TU Zhui, HE Qing-hua. Research on Panning Methods of Single-Domain Heavy-Chain Antibody Fragments for Aflatoxin B1 from Non-Immune Library[J]. Journal of Food Science and Biotechnology,2011,30(6):950-955.

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在线发布日期: 2014-06-17
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