运用噬菌体随机肽库筛选与DON毒素特异结合的亲和肽段  

High-affinity peptides binding to deoxynevalenol screened from phage display peptide library

作　　者：邢锦城[1,2] 张旭[1] 马鸿翔[1] 徐海[3] 侯继波[3] 

机构地区：[1]江苏省农业科学院,江苏省农业生物学重点实验室,南京210014 [2]江苏沿海地区农业科学研究所,盐城224002 [3]江苏省农业科学院,国家兽用生物制品工程技术研究中心,南京210014

出　　处：《植物保护学报》2015年第2期194-200,共7页Journal of Plant Protection

基　　金：国家公益性行业(农业)科研专项(201303016);江苏省科技支撑项目(BE2012306);江苏省自主创新项目(CX(11)4035)

摘　　要：为获得与毒素脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)高亲和力的特异性多肽,利用丁基硼酸封闭法制备DON人工抗原DON-HG-BSA,以其为靶分子筛选噬菌体肽库,对阳性克隆进行DNA序列测定与氨基酸推导,并进行亲和特异性验证。结果显示,经过4轮亲和肽段筛选与酶联免疫检测法鉴定,从M13噬菌体随机7肽库中得到15个DON人工抗原包被孔与牛血清白蛋白包被孔OD比值大于3.8的阳性克隆,其中10个与DON人工抗原高亲和力结合的阳性克隆经测序后共推导出4种多肽序列。经抗原梯度酶联免疫检测法验证,多肽MAPGWVP与DON人工抗原浓度的相关性高达0.99,对人工抗原具有高亲和力。该多肽的获得将为进一步开发高效的DON诊断试剂盒奠定基础。To obtain specific polypeptides that bind to mycotoxin deoxynivalenol( DON) with high affinity,DON was converted to 3-HG-DON after protection of the C7- and C15-hydroxyls with a cyclic boronate ester,and then conjugated to bovine serum albumin( BSA) to make artificial antigen DON-HGBSA. The Ph. D.TM-7 phage display peptide library was screened by DON-HG-BSA and the positive clones were sequenced to deduce the core peptides. After four rounds of biopanning,15 positive clones,of which the OD ratio of coated antigen to coated BSA was greater than 3. 8,were selected by enzymelinked immunosorbent assay( ELISA). Gene sequencing showed that ten selected positive clones encoded four peptides. ELISA analysis indicated that the core peptide MAPGWVP had the highest affinity to the antigen with a correlation coefficient of 0. 99. The peptides with high affinity to DON could be obtained from phage display random peptide library,which provides a potential tool for developing highly sensitive DON diagnostic kits.

关 键 词：脱氧雪腐镰刀菌烯醇(DON) 噬菌体展示随机肽库 酶联免疫检测 小麦赤霉病 

分 类 号：S432[农业科学—植物病理学]