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