Expression of the soybean allergenic protein P34 in Escherichia coli and its indirect ELISA detection method
Expression of the soybean allergenic protein P34 in Escherichia coli and its indirect ELISA detection method
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DOI：
10.1007/s00253-012-4006-3
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发表时间：
2012-06-01
影响因子：
5
通讯作者：
Wang, Jianhua
中科院分区：
文献类型：
--
作者：
Liu, Bin;Teng, Da;Wang, Jianhua
为了检测大豆制品中的大豆过敏原P34（Bd 30K），合成了P34的全长cDNA序列，并将其插入原核表达载体pET-28a中。在0.8 mmol/L异丙基β-1-硫代半乳糖苷的诱导下，P34蛋白在BL21（DE3）中以包涵体形式表达。经过His-Bind亲和层析纯化后，重组蛋白纯度超过92%，其分子量（约33 kDa）与天然大豆P34非常接近。用纯化的重组P34制备抗P34的多克隆抗体(pAB)。生成的 pAB 命名为 pAB-P34，对豆粕中的 P34 蛋白表现出高度特异性。建立了基于pAB-P34的间接酶联免疫吸附法（iELISA）测定豆制品中P34的含量。 P34回收率试验的CV小于7.77%，表明iELISA具有较高的重现性和准确性。因此，表达系统中产生的重组P34、制备的pAB-P34以及开发的iELISA可以为各种豆制品中P34的灵敏检测以及未来与大豆P34相关的过敏研究提供有价值的工具。
To detect the soybean allergen P34 ( 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 BL21 (DE3) as an inclusion body under the induction of 0.8 mmol/L isopropyl beta--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 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.