Expression of the soybean allergenic protein P34 in Escherichia coli, its indirect ELISA detection method

摘要

To detect the soybean allergen P34 (Gly m Bd 30K) from soybean products, the full-length cDNA sequence of P34 was synthesized, inserted into the prokaryotic expression vector pET-28a. The P34 protein was expressed in Escherichia coli BL21 (DE3) as an inclusion body under the induction of 0.8 mmol/L isopropyl β-D-1-thiogalactopyranoside. After purification with His-Bind affinity chromatography, the purity quotient of the recombinant protein was over 92%, its molecular weight (approximately 33 kDa) was very close to that of the native soybean P34. The polyclonal antibody (pAB) against P34 was prepared with the purified recombinant P34. The generated pAB, named as pAB-P34, exhibited high specificity to the P34 protein of the soybean meal. The indirect enzyme-linked immunosorbent assay (iELISA) based on pAB-P34 was established to determine the P34 content of soybean products. The CVs of the recovery tests of P34 were less than 7.77%, which indicated that iELISA had high reproducibility, accuracy. Therefore, the recombinant P34 produced in the E. coli expression system, the prepared pAB-P34, the developed iELISA could provide a valuable tool for sensitive detection of P34 in various soybean products, for future studies on allergies related to soybean P34.