| 抗黄曲霉毒素单链抗体基因的克隆与表达 |
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| 引用本文： | 李鑫,李培武,张奇,张文,李园园.抗黄曲霉毒素单链抗体基因的克隆与表达[J].中国油料,2012(5):528-532. |
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| 作者姓名： | 李鑫 李培武 张奇 张文 李园园 |
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| 作者单位： | [1]中国农业科学院油料作物研究所,湖北武汉430062 [2]农业部油料作物生物学与遗传育种重点实验室,湖北武汉430062 [3]农业部生物毒素检测重点实验室,湖北武汉430062 [4]农业部油料产品质量安全风险评估实验室（武汉）,湖北武汉430062 [5]农业部油料及制品质量监督检验测试中心,湖北武汉430062 |
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| 基金项目： | 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） |
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| 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。
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| 关 键 词： | 黄曲霉毒素 抗体可变区 单链抗体 |
Cloning and expression of single chain Fv antibody gene against aflatoxin |
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| Authors: | LI Xin LI Pei-wu ZHANG Qi ZHANG Wen LI Yuan-yuan |
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| Affiliation: | 1.Oil Crops Research Institute of Chinese Academy of Agricultural Sciences,Wuhan 430062,China; 2.Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture,Wuhan 430062,China; 3.Key Laboratory of Detection for Mycotoxins,Ministry of Agriculture,Wuhan 430062,China; 4.Laboratory of Risk Assessment for Oilseeds Products(Wuhan),Ministry of Agriculture,Wuhan 430062,China; 5.Quality Inspection and Test Center for Oilseeds Products,Ministry of Agriculture,Wuhan 430062,China) |
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| Abstract: | 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. |
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| Keywords: | Aflatoxin Variable region genes Single-chain Fv（ScFv） |
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