摘要
为降低黄曲霉毒素抗体制备成本,在已有的能分泌抗黄曲霉毒素单克隆抗体的杂交瘤细胞系8F6的基础上,成功克隆得到了该单克隆抗体的重链(VH)和轻链可变区(VL)基因片段。通过重叠延伸PCR的方法将轻、重链可变区基因连接,并引入连接肽(Linker)编码序列,构建VH-Linker-VL结构的单链抗体(ScFv)基因,并将该基因克隆到噬菌体表达载体pCANTAB 5E上,使单链抗体以噬菌体展示形式在大肠杆菌TG1中表达。间接竞争ELISA方法检测到该ScFv对黄曲霉毒素B1的抑制率(IC50)值为0.57ng/mL,表明该单链抗体与亲本鼠单抗有相同的抗原结合特异性,且具有很高灵敏度。
The objective of this study was to reduce the preparation cost of aflatoxin antibody.Based on the hybridoma cell 8F6 which secrete mAbs against aflatoxin,the variable heavy（VH） and light（VL） region genes of 8F6 were amplified using polymerase chain reaction.The VH-linker-VL genes were constructed by overlap extension,then cloned into a phagemid vector pCANTAB 5E and expressed in E.coli TG1 cells as a fusion with a phage protein.Indirect competitive ELISA was performed for detection of the binding activity,which resulted in IC50 value of the constructed ScFv as 0.57ng per milliliter.
出处
《中国油料作物学报》
CAS
CSCD
北大核心
2012年第5期528-532,共5页
Chinese Journal of Oil Crop Sciences
基金
农业部948项目(2010-G1
2011-G5)
国家自然科学基金(31171702
31101299)
国家农业行业科技专项(201203094
201203037)
国家科技支撑计划(2012BAB19B09
2012BAK08B03)
中国农业科学院油料作物研究所所长基金项目(1610172010003)
关键词
黄曲霉毒素
抗体可变区
单链抗体
Aflatoxin
Variable region genes
Single-chain Fv（ScFv）
