苦荞10 kD过敏原的重组表达及部分性质分析

doi:10.7506/spkx1002-6630-201403026

摘要/Abstract

摘要：

以苦荞种子灌浆期cDNA文库中获得的苦荞10 kD过敏原基因序列TBAP10（tartary buckwheat 10 kD allergenprotein，TBAP10；GenBank登录号JK729379.1）为基础，构建重组表达载体pET47b-TBAP10，重组蛋白在大肠杆菌BL21 Star（DE3）中以包涵体形式表达。经包涵体复性和钴离子螯合层析纯化目的蛋白，并对其过敏活性、热稳定性及在模拟胃肠环境中的稳定性进行了分析。Western blotting显示该蛋白与苦荞16 kD过敏蛋白Fag t2存在免疫交叉反应。竞争性ELISA证明重组蛋白TBAP10具有与荞麦过敏患者血清IgE特异的结合活性；TBAP10具有强的热稳定性，能耐受15 min的沸水浴；模拟胃肠环境的消化结果显示TBAP10对胃蛋白酶具有强的耐受性，但对胰蛋白酶无耐受性。

关键词: 原核表达, 过敏蛋白, 免疫性质, 模拟胃肠消化

Abstract:

The 10 kD allergen gene (TBAP10) of tartary buckwheat was obtained from the full-length cDNA library during
the seed-filling period, and a prokaryotic recombinant expression vector pET47b inserted with TBAP10 gene (pET47b-
TBAP10) was constructed. The target protein TBAP10 was expressed in E. coli BL21 Star (DE3) as an inclusion body
form. After re-folding and purification by cobalt ion chelating affinity chromatography of the isolated inclusion body,
allergenic activity, thermal stability and stability in simulated gastrointestinal environment of the purified TBAP10 protein
were analyzed. Western blotting revealed the immunogenic cross-reactivity between 16 kD buckwheat allergic protein and Fag t2.
Competitive ELISA demonstrated that the recombinant protein had a binding capability to IgE from buckwheat-allergic
patients. TBAP10 had strong thermal stability and could withstand boiling water bath for 15 min. Simulated gastrointestinal
digestion experiments indicated that the recombinant protein had strong tolerance to pepsin, while it could be totally
degraded after trypsin digestion.

Key words: overexpression, allergenic protein, immunological properties, simulated gastrointestinal digestion

陈鹏，封雪，王磊，邓丹丹，李学俊. 苦荞10 kD过敏原的重组表达及部分性质分析[J]. 食品科学, doi: 10.7506/spkx1002-6630-201403026.

CHEN Peng, FENG Xue, WANG Lei, DENG Dan-dan, LI Xue-jun. Overexpression and Characterization of 10 kD Allergenic Protein in Tartary Buckwheat[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201403026.

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苦荞10 kD过敏原的重组表达及部分性质分析

Overexpression and Characterization of 10 kD Allergenic Protein in Tartary Buckwheat

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