高等学校化学学报

高等学校化学学报 ›› 2012, Vol. 33 ›› Issue (10): 2269.doi: 10.7503/cjcu20120158

• 生物化学 • 上一篇    下一篇

二乙氧基硫代磷酸酯类有机磷农药单链抗体的制备及广谱特异性

周丽君1, 黄启欣2, 雷红涛2, 王欲晓1, 王强2, 徐振林2, 王弘2, 孙远明2   

  1. 1. 解放军海军总医院中心实验科, 北京 100048;
    2. 广东省食品质量安全重点实验室, 华南农业大学食品学院, 广州 510640
  • 收稿日期:2012-02-28 出版日期:2012-10-10 发布日期:2012-09-12
  • 通讯作者: 孙远明, 男, 教授, 主要从事食品质量与安全方面的研究. E-mail: ymsun@scau.edu.cn E-mail:ymsun@scau.edu.cn
  • 基金资助:

    国家"九七三"计划项目(批准号: 2012CB720803); 国家自然科学基金(批准号: 30700663); 高等学校博士点基金项目(批准号: 20114404130002)和广州市科技计划项目(批准号: 12C12101663)资助.

Preparation of a Single-chain Variable Fragment for O,O-Diethyl Phosphorothioate Pesticides and Its Broad-specificity

ZHOU Li-Jun1, HUANG Qi-Xin2, LEI Hong-Tao2, WANG Yu-Xiao1, WANG Qiang2, XU Zhen-Lin2, WANG Hong2, SUN Yuan-Ming2   

  1. 1. Central Laboratory of Navy General Hospital, Beijing 100048, China;
    2. Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510640, China
  • Received:2012-02-28 Online:2012-10-10 Published:2012-09-12

PDF (PC)

被引次数

10

摘要：

从分泌抗二乙氧基硫代磷酸酯类有机磷农药(DPPs)单克隆抗体(MAb)的杂交瘤细胞系(12C2)中提取了总RNA, 经RT-PCR反转录成cDNA, 设计带linker引物, 采用重叠延伸PCR制备单链抗体(scFv)基因, 将其克隆到噬菌体载体p3MH中, 构建成噬菌体单链抗体表达载体, 转化大肠杆菌表达出噬菌体表面展示scFv, 对经过Phage-ELISA鉴定的阳性克隆进行噬菌体外壳蛋白基因geneⅢ的去除, 用IPTG诱导其可溶性表达, 对表达产物进行SDS-PAGE, Western-Blot及ELISA鉴定, 并与亲本MAb进行性能对比. 结果表明, 可溶性表达的scFv分子量为27000; scFv与DPPs的交叉反应率比其亲本MAb提高了1.3~3.5倍, 表明其广谱特异性有所提高. 由于scFv与MAb相比具有诸多优点, 因此本研究为有机磷农药多残留检测方法的建立提供了一种更广谱、 更灵敏的新型识别分子.

关键词: 二乙氧基硫代磷酸酯类有机磷农药, 单链抗体, 噬菌体展示, 广谱特异性

Abstract:

The total RNA was extracted from a hybridoma cell line(12C2) secreting monoclonal antibodies(MAb) against O,O-diethyl phosphorothioate pesticides(DPPs) and reverse-transcribed into cDNA by RT-PCR. The scFv was amplified by gene splicing via overlap extension PCR(SOE-PCR) with the previously designed degenerate primers with a DNA linker encoding(G1y4Ser)3. Then the scFv fragment was cloned into the phagemid p3MH and then the phagemid was transformed into the competent Escherichia coli XL1-Blue. With the rescue of helper phage VCSM13, a phage-display scFv was constructed and then characterized by phage-ELISA. Furthermore, a soluble scFv was gained by wiping off the gene Ⅲ on p3MH then induced by IPTG. After the detection of SDS-PAGE, Western-Blot and ELISA, characteristics of scFv were compared with those of parental MAb. With a molecular weight of 27000, the DPPs scFv owns a broader specificity and a higher sensitivity than MAb. The IC50 and LOD to detected DPPs enhanced 1.3-3.5 times of parental MAb. This research will provide a new broad-specific and high-sensitive recognition molecule to the detection for DPPs.

Key words: O,O-Diethyl phosphorothioate pesticides, Single chain Fv(scFv), Phage-display, Broad-specificity

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