抗黄曲霉毒素单链抗体基因的克隆与表达

Cloning and expression of single chain Fv antibody gene against aflatoxin

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.