摘要
根据抗玉米赤霉烯酮毒素单链抗体的氨基酸序列及大肠杆菌偏爱密码子,用重叠延伸PCR法合成了单链抗体的重链和轻链,经(Gly4Ser)2Linker连接,获得完整的单链抗体基因ZEN2 scFv。将ZEN2 scFv与碱性磷酸酶(AP)编码序列连接形成融合蛋白基因ZEN2 scFv-AP,构建到pET载体,转化大肠杆菌菌株BL21(DE3),经IPTG诱导表达及亲和层析纯化,在SDS-PAGE和Western blot分析中均检测到1条分子质量为75 ku的可溶性蛋白条带,ELISA分析证实,细菌表达的ZEN2 scFv-AP融合蛋白具有碱性磷酸酶活性。这一研究结果为建立快速、灵敏、经济的玉米赤霉烯酮毒素的ELISA检测奠定了基础。
Based on the amino acid sequence of a single-chain antibody specific for zearalenone mycotoxin and codon usage in E. coli, a variable heavy chain （VH） and a variable light chain （VL） were synthesized by PCR to create a single-chain （scFv） antibody gene, ZEN2, that was linked by a （Gly4Ser）2 linker. The scFv antibody gene, ZEN2, was further ligated into an alkaline phosphatase （AP） sequence to construct a fusion protein, ZEN2 scFv-AP that was cloned into pET vector. E. coli strain BL21 （DE3） was transformed with the pET plasmid containing ZEN2 scFv-AP and used for the expression of the fusion protein upon induction by isopropylthio-β-D-galactoside （IPTG）. The bacterially expressed soluble fusion protein was purified by affinity chromatography. SDS-PAGE and Western blot analyses of the purified protein detected a 75 ku protein band. Enzyme-linked immunosorbent assay （ELISA） revealed an enzymatic activity for the alkaline phosphatase in the fusion protein ZEN2 scFv-AP. These results would serve as a foundation for the development of a rapid, accurate and cheap method for the detection of zearalenone mycotoxin via ELISA.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2008年第6期697-700,共4页
Journal of Huazhong Agricultural University
基金
国家"863"高技术研究发展专项(2007AA10Z425)
国家自然科学基金项目(30530510
30771337)资助
关键词
玉米赤霉烯酮
单链抗体
碱性磷酸酶
融合蛋白
细菌表达
zearalenone single-chain antibody alkaline phosphatase fusion proteins bacterial expression
作者简介
李鑫，男，1981年生，华中农业大学生命科学技术学院硕士研究生，武汉430070
通讯作者E-mail：hepingli@mail．hzau．edu．cn
