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  

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

作　　者：周丽君 黄启欣[2] 雷红涛[2] 王欲晓 王强[2] 徐振林[2] 王弘[2] 孙远明[2] 

机构地区：[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

出　　处：《高等学校化学学报》2012年第10期2269-2274,共6页Chemical Journal of Chinese Universities

基　　金：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);广州市科技计划项目(批准号:12C12101663)资助

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.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 de- signed degenerate primers with a DNA linker encoding（Gly4Ser） 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.

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 

分 类 号：O652[理学—分析化学]