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马巍娜, 贾雷立, 刘雪林, 宋宏彬, 高志贤, 钟彦伟, 张传福, 徐元勇. 抗盐酸克伦特罗人源ScFv抗体筛选与鉴定[J]. 中国公共卫生, 2009, 25(3): 287-288. DOI: 10.11847/zgggws2009-25-03-18
引用本文: 马巍娜, 贾雷立, 刘雪林, 宋宏彬, 高志贤, 钟彦伟, 张传福, 徐元勇. 抗盐酸克伦特罗人源ScFv抗体筛选与鉴定[J]. 中国公共卫生, 2009, 25(3): 287-288. DOI: 10.11847/zgggws2009-25-03-18
MA Wei-na, JIA Lei-li, LIU Xue-lin, . Screening and characterization of human phage antibody to clenbuterol(CLB)[J]. Chinese Journal of Public Health, 2009, 25(3): 287-288. DOI: 10.11847/zgggws2009-25-03-18
Citation: MA Wei-na, JIA Lei-li, LIU Xue-lin, . Screening and characterization of human phage antibody to clenbuterol(CLB)[J]. Chinese Journal of Public Health, 2009, 25(3): 287-288. DOI: 10.11847/zgggws2009-25-03-18

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)

  • 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-Blue,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。

     

    Abstract: 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 humanan tibody positive bacteri-ophage were identified with Sfi I/Not I enzyme digestion and subcloned into pCANAB 5E vector and transfered into E.coli XL1-Blue.DNA sequences of the bacteriophage selected and encoding sequences of CLBS cFv 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 EU 681272.Conclusion An initial CLB of the anti-ScFv antibody was acquired with phage display technology and the experiment laid a basis for further study.

     

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