摘要
【目的】从Tomlinson(I)噬菌体抗体库中筛选人源化抗黄曲霉毒素B1单链抗体蛋白(scFv)并进行鉴定。【方法】分别采用甘氨酸洗脱、胰蛋白酶洗脱、游离AFB1竞争洗脱和AFB1竞争洗脱加胰蛋白酶处理4种方法对噬菌体抗体进行特异性洗脱。将筛选到的噬菌体阳性克隆转化到大肠杆菌(Escherichia coli.)HB2151,IPTG诱导表达scFv。ELISA检测和基因序列测定。【结果】比较4种洗脱方法,发现用AFB1竞争加胰蛋白酶洗脱筛选到阳性克隆的的概率最高,把此方法得到的阳性噬菌体克隆转化大肠杆菌HB2151表达,竞争性ELISA检测得到2个能特异性结合游离的AFB1阳性克隆。间接性ELISA测定相对亲和力分别为0.4μg/mL和0.7μg/mL。测序证实scFv属于人类免疫球蛋白可变区。【结论】利用噬菌体展示技术获得高特异性抗黄曲霉毒素B1的人源化单链抗体,本实验方法可以为其它抗半抗原重组抗体的筛选提供一定的借鉴意义。
[ Objective] To screen and identify anti- Aflatoxin B1 single chain antibodies （scFv） from Tomlinson （I） library. [Methods] The phages absorbed on the ELISA plate were eluted by four methods including glycine buffer elution, trypsin elution, AFB1 competitive elution and competitive elution following trypsin treatment. The phage positive clones were transformed into Eseherichia coli HB2151 and soluble scFv protein was expressed with the induction of Isopropyl β-D-1-Thiogalavtopy ranoside （IPTG）. The seFv was identified by ELISA and DNA sequence. [Results] Comparing the four methods, we found that the most efficient way to get positive clones was competitive elution following trypsin treatment. Obtainning two positive clones that could bind specifically with free AFB1 , which their relative affinity were 0.4 μg/mL and 0.7 μg/mL, respectively. DNA sequencing results showed that the scFv belonged to human immunoglobulin variable region. [ Conclusion] The specific human seFv could be obtained with phage antibody library, and our method provided an alternative for screening recombinant antibody against anti- hapten.
出处
《微生物学报》
CAS
CSCD
北大核心
2009年第1期135-140,共6页
Acta Microbiologica Sinica
基金
教育部新世纪优秀人才支持计划资助项目(NCET-06-0482)~~
关键词
黄曲霉毒素B1
重组抗体
筛选
鉴定
aflatoxin
recombinant antibody
screening
identification
