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

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.

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

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.