| 抗克伦特罗单链抗体表达载体的构建与鉴定 |
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| 引用本文： | 王弘,梁艳,杨金易,刘细霞,张宏斌,雷红涛,沈玉栋,孙远明.抗克伦特罗单链抗体表达载体的构建与鉴定[J].生物工程学报,2008,24(8):1470-1474. |
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| 作者姓名： | 王弘 梁艳 杨金易 刘细霞 张宏斌 雷红涛 沈玉栋 孙远明 |
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| 作者单位： | 1. 广东省高等学校食品质量安全重点实验室/华南农业大学食品质量安全研究所,广州,510640
2. 广州军区广州总医院医学实验中心,广州,510010 |
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| 基金项目： | 国家自然科学基金资助 (Nos. 30400354 and 20477012), 国家“863”计划 (No. 2007AA10Z437), 广东省自然科学基金 (No. 06025825), 高等学校博士点基金 (No. 20050564011)资助。 |
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| 摘 要： | 为构建克伦特罗(Clenbuterol, CBL)单链抗体(scFv)表达载体, 实现其在大肠杆菌中的表达。以pCANTAB5E- CBL质粒为模板, 扩增CBL的scFv基因, 与pPICZaA载体重组, 然后以重组质粒pPICZaA-scFv为模板扩增scFv及其相连的6 个组氨酸(6×His)片段, 再与载体pBV220连接, 转化大肠杆菌DH5a, 阳性克隆质粒经酶切和PCR鉴定后进行目的片断的序列测定。重组菌经温度诱导表达重组单链抗体, 并通过SDS-PAGE和Western blotting对其鉴定。采用竞争ELISA检测重组单链抗体的抗原结合活性。结果表明重组质粒pBV220-scFv含有插入片段, 与原序列的同源性达99.8%。重组蛋白的分子量接近37 kD, 能够被抗His标签单克隆抗体特异性识别且可被游离的CBL竞争性抑制, IC50值为4.55 ng/mL。这说明我们成功构建了重组质粒pBV220-scFv并实现了其在大肠杆菌中的表达, 为进一步进行CBL免疫法快速检测奠定了一定的基础。
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| 关 键 词： | 克伦特罗 重组单链抗体 原核表达 |
| 收稿时间： | 2007/12/11 0:00:00 |
Construction and Expression of Anti-clenbuterol Single Chain Fv Recombinant Vector |
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| Hong Wang,Yan Liang,Jingyi Yang,Xixia Liu,Hongbin Zhang,Hongtao Lei,Yudong Shen and Yuanming Sun.Construction and Expression of Anti-clenbuterol Single Chain Fv Recombinant Vector[J].Chinese Journal of Biotechnology,2008,24(8):1470-1474. |
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| Authors: | Hong Wang Yan Liang Jingyi Yang Xixia Liu Hongbin Zhang Hongtao Lei Yudong Shen and Yuanming Sun |
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| Affiliation: | The Higher Education Key Laboratory of Food Quality and Safety of Guangdong Province /Research Institute of Food Quality and Safety of SCAU, South China Agricultural University, Guangzhou 510640, China;The Higher Education Key Laboratory of Food Quality and Safety of Guangdong Province /Research Institute of Food Quality and Safety of SCAU, South China Agricultural University, Guangzhou 510640, China;The Higher Education Key Laboratory of Food Quality and Safety of Guangdong Province /Research Institute of Food Quality and Safety of SCAU, South China Agricultural University, Guangzhou 510640, China;The Higher Education Key Laboratory of Food Quality and Safety of Guangdong Province /Research Institute of Food Quality and Safety of SCAU, South China Agricultural University, Guangzhou 510640, China;Guangzhou Army General Hospital, Guangzhou 510010, China;The Higher Education Key Laboratory of Food Quality and Safety of Guangdong Province /Research Institute of Food Quality and Safety of SCAU, South China Agricultural University, Guangzhou 510640, China;The Higher Education Key Laboratory of Food Quality and Safety of Guangdong Province /Research Institute of Food Quality and Safety of SCAU, South China Agricultural University, Guangzhou 510640, China;The Higher Education Key Laboratory of Food Quality and Safety of Guangdong Province /Research Institute of Food Quality and Safety of SCAU, South China Agricultural University, Guangzhou 510640, China |
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| Abstract: | |
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| Keywords: | clenbuterol (CBL) recombinant scFv prokaryotic expression |
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