Lipocalin文库的构建及与克百威有结合活性的小分子筛选  

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

作　　者：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] 

机构地区：[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

出　　处：《高等学校化学学报》2008年第3期528-532,共5页Chemical Journal of Chinese Universities

基　　金：国家“八六三”计划(批准号:2006AA10Z447);国家自然科学基金(批准号:20477012,30400354);高等学校博士学科点基金(批准号:20050564011);广州市科技攻关重点项目(批准号:2004Z2-E0031);粤港关键领域重点突破招标项目(批准号:2006A25005002)资助

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K7anticalin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.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 sensi- tive 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 pCANTABSE was used to construct the lipocalin library. The library repotire is 4.0 × 10^9. 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. Dotblotting showed that phage displayed anticalin, named as KT, bound carbofuran specifically among them. K7 can be used as potential anticalin to detect carbofuran of crops.

关 键 词：Lipocalin文库 克百威 Anticalin分子 

分 类 号：Q514[生物学—生物化学]