title: Expression of the soybean allergenic protein P34 in Escherichia coli and its indirect ELISA detection method.
abstract: To detect the soybean allergen P34 (Gly m Bd 30K) from soybean products, the full-length cDNA sequence of P34 was synthesized and 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 %, and 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 and accuracy. Therefore, the recombinant P34 produced in the E. coli expression system, the prepared pAB-P34, and the developed iELISA could provide a valuable tool for sensitive detection of P34 in various soybean products and for future studies on allergies related to soybean P34.
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, People's Republic China.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.forename: Da
authors.initials: D
authors.lastname: Teng
authors.forename: Xiumin
authors.initials: X
authors.lastname: Wang
authors.forename: Yalin
authors.initials: Y
authors.lastname: Yang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wang
mesh_terms: D000485:Allergens / Q000032:analysis* / Q000276:immunology; D000906:Antibodies; D052179:Antigens, Plant; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005504:Food Analysis / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D030262:Soybean Proteins / Q000032:analysis* / Q000276:immunology; D013025:Glycine max / Q000235:genetics
chemical_list: D000485:Allergens; D000906:Antibodies; D052179:Antigens, Plant; D011994:Recombinant Proteins; D030262:Soybean Proteins; C075135:P34 protein, Glycine max