Lipocalin文库的构建及与克百威有结合活性的小分子筛选

高等学校化学学报

Lipocalin文库的构建及与克百威有结合活性的小分子筛选

武婕^1,2, 杨金易¹, 张宏斌², 潘科¹, 王弘¹, 雷红涛¹, 郑霖¹, 肖治理¹, 孙远明¹

1. 广东省高等学校食品质量安全重点实验室, 华南农业大学食品质量与安全研究所, 广州 510640;
2. 广州军区广州总医院医学试验科, 广州 510010

收稿日期:2007-06-18 修回日期:1900-01-01 出版日期:2008-03-10 发布日期:2008-03-10
通讯作者: 孙远明

Construction of Phage-displayed Library Based on the Lipocalin Scaffold and Screening Anticalins with Specificity for Carbofuran

WU Jie^1,2, YANG Jing-Yi¹, ZHANG Hong-Bin², PAN Ke¹,WANG Hong¹, LEI Hong-Tao¹, ZHENG Lin¹, XIAO Zhi-Li¹, SUN Yuan-Ming¹*

1. The Higher Education Key Laboratory of Food Quality and Safety of Guangdong Province, Research Institute of Food Quality and Safety of SCAU, South China Agricultural University, Guangzhou 510640, China;
2. Department of Medical Research, Guangzhou Army General Hospital, Guangzhou 510010, China

Received:2007-06-18 Revised:1900-01-01 Online:2008-03-10 Published:2008-03-10
Contact: SUN Yuan-Ming

摘要/Abstract

摘要： 构建大菜粉蝶Lipocalin蛋白家族中的BBP蛋白突变文库, 依据基因序列, 分别设计10条和12条引物, 通过PCR重叠延伸法得到包含随机突变蛋白BBP的基因序列, 并重组到噬菌粒载体pCANTAB5E中构建Lipocalin突变文库, 库容达到4.0×10⁹. 以偶联分子BSA-克百威和OVA-克百威为靶分子, 采用柱式和平皿式交叉法对Lipocalin文库进行了筛选, 用竞争洗脱法洗脱特异结合活性的噬菌体. 经过3轮筛选, 从第3轮的洗脱液中随机挑选了10个重组克隆, 用Dot-blotting法检测出K7 anticalin分子能与克百威特异结合. 为研发克百威的Anticalin类检测试剂盒提供了候选分子, 也为拓宽Lipocalin文库在有害小分子检测方面的应用奠定了基础.

关键词: Lipocalin文库, 克百威, Anticalin分子

Abstract: Carbofuran, which is used as toxic carbonate pesticide of crops, is highly toxic and small molecule. To detect the remainder of carbofuran efficiently is important to our healthy. The lipocalins, a family of diverse proteins that normally serve for the storage or transport of mostly hydrophobin and/or chemically sensitive organic compounds, can be reshaped by combinatorial protein design such that it recognizes small molecules. Sixteen residues at the center of binding site of the bilin-binding protein(BBP) from Pieris brassicae were subjected to random mutagenesis by overlapping PCR with 12 primers. Phagemid expression vector of pCANTAB5E was used to construct the lipocalin library. The library repotire is4.0×10⁹. BSA and OVA conjugated carbofuran respectively as the targeted protein, the library was screened with column and plate as supporter. After 3 rounds of panning, 10 phage clones were random selected with competitive elution. Dot-blotting showed that phage displayed anticalin, named as K7, bound carbofuran specifically among them. K7 can be used as potential anticalin to detect carbofuran of crops.

Key words: Lipocalin library, Carbofuran, Anticalin molecule

中图分类号:

Q514

TrendMD:

武婕,杨金易,张宏斌,潘科,王弘,雷红涛,郑霖,肖治理,孙远明 . Lipocalin文库的构建及与克百威有结合活性的小分子筛选. 高等学校化学学报, doi: .

WU Jie^1,2, YANG Jing-Yi¹, ZHANG Hong-Bin², PAN Ke¹,WANG Hong¹, LEI Hong-Tao¹, ZHENG Lin¹, XIAO Zhi-Li¹, SUN Yuan-Ming¹*. Construction of Phage-displayed Library Based on the Lipocalin Scaffold and Screening Anticalins with Specificity for Carbofuran. Chem. J. Chinese Universities, doi: .