Expression, purification of codon-optimized ochratoxin A nanobody-GST fusion protein and its one-step immunoassay for detection of OTA in cereal

Abstract

The ochratoxin A (OTA) nanobody-Glutathione S-transferase (GST) fusion protein preparation and one-step immunoassay for detection OTA in cereal were described in this study. We optimized the OTA nanobody codons and the soluble OTA nanobody-GST fusion protein was expressed in Escherichia coli (E. coli). The expression conditions were optimized and the high-yield fusion protein was purified by GST affinity method. Then, the one-step immunoassay was selected to detect cereal samples by a comparison among three immunoassay modes. SDS-PAGE and western blot analyses revealed that the molecular weight of OTA nanobody-GST fusion protein was about 41 kDa. The expression yield reached to 32.9%, and the purified fusion protein was 155 mg/L bacteria. The one-step immunoassay indicated that the average concentration required for 50% inhibition of binding (IC50) and the limit of detection (LOD) for OTA were 2.25 ± 0.08 ng/mL and 0.32 ± 0.06 ng/mL, and the linear range was from 0.63 ± 0.08 ng/mL to 10.62 ± 1.27 ng/mL. The recovery rate of spiked cereal was from 85.34% to 116.19%. The present findings indicated that the entire time of one-step immunoassay was 15 min. It could be further used to develop a rapid detection kit for detection of OTA.