抗盐酸克伦特罗人源ScFv抗体筛选与鉴定  

Screening and characterization of human phage antibody to clenbuterol(CLB)

作　　者：马巍娜[1] 贾雷立[1] 刘雪林[1] 宋宏彬[1] 高志贤[2] 钟彦伟[3] 张传福[1] 徐元勇[1] 

机构地区：[1]中国人民解放军疾病预防控制所卫生学评价中心,北京100071 [2]中国人民解放军军事医学科学院卫生学环境医学研究所 [3]中国人民解放军第302医院传染病研究所基因治疗研究中心

出　　处：《中国公共卫生》2009年第3期287-288,共2页Chinese Journal of Public Health

基　　金：国家科技支撑计划基金资助(2006BAK02A09);国家自然科学基金(30671927,30772001)

摘　　要：目的筛选盐酸克伦特罗(CLB)人源单链可变区(ScFv)抗体,为研究快速检测试剂(盒)提供基础依据。方法采用噬菌体表面展示技术,以CLB作为抗原,从噬菌体单链可变区抗体库中经过3轮"吸附-洗脱-扩增"筛选过程,随机挑选抗CLB的60个克隆,利用酶联免疫吸附法(ELISA)、交叉反应及竞争抑制实验,对其进行免疫学检测和鉴定,获得与CLB结合活性较强的ScFv阳性克隆,从噬菌体抗体阳性克隆中提取质粒,经酶切鉴定SfiⅠ/NotⅠ后,亚克隆到pCANTAB5E载体;转化大肠埃希菌XL1-B lue,提取质粒进行DNA序列测定;并对CLB ScFv的编码基因序列分析。结果经过筛选60个克隆中有15株克隆ELISA的吸光度(A490nm)值较高,将这些噬菌体上清与牛血清白蛋白(BSA)进行交叉反应,确定有7株交叉反应较弱,结合3次ELISA重复实验的A值及竞争抑制实验结果,最后确定1株阳性克隆。亚克隆到pCANTAB5E载体;转化大肠埃希菌XL1-B lue,提取质粒进行DNA序列测定;DNA为750 bp;经BLAST分析,此片段属于新抗体基因序列,GenBank注册号为:EU681272。结论本实验用噬菌体抗体库技术能够初步获得抗CLB的ScFv抗体,为下一步其特异性亲和力的研究创造了条件。Objective To screen the clenbuterol （CLB） humanized single-chain variable region （ScFv） antibody for the study of rapid detection kit. Methods Sixty anti-CLB clones were randomly selected from the phage single-chain variable region（ScFv） antibody libary through adsorbtion-elution-amplification procedures with phage display technology. The immunity of the clones was tested with enzyme-linked immunosorbent assay （ELISA） and cross-reaction suppression test and clones with strong positive immune activity were select. The plasmids from the selected human antibody positive bacteri- ophage were identified with Sfi I/Not I enzyme digestion and subcloned into pCANAB5E vector and transfered into E. coli XL1 - Blue. DNA sequences of the bacteriophage selected and encoding sequences of CLB ScFv were analyzed. Results Based on the results of repeated ELISA tests and cross-reaction suppression test one positive clone was identified with new antibody fragment gene sequence of 750 bp and a GenBank registration number EU681272. Conclusion An initial CLB of the anti-ScFv antibody was acquired with phage display technology and the experiment laid a basis for further study.

关 键 词：噬菌体表面展示技术 盐酸克伦特罗 人源单链可变区抗体(ScFv) 

分 类 号：R392.11[医药卫生—免疫学]