AM79 EPSPS蛋白的体外模拟胃肠液消化稳定性研究

doi:10.13560/j.cnki.biotech.bull.1985.2017-0072

生物技术通报 ›› 2017, Vol. 33 ›› Issue (6): 176-181.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0072

AM79 EPSPS蛋白的体外模拟胃肠液消化稳定性研究

王玉^1,2, 王建华¹, 刘允军²

1. 中国农业大学农学院,北京 100094;
2.中国农业科学院作物科学研究所,北京 100081

收稿日期:2017-02-10 出版日期:2017-06-26 发布日期:2017-06-19
作者简介:王玉,女,硕士,研究方向：分子生物学与转基因检测;E-mail：llcwangy1103@163.com
基金资助:
转基因新品种培育重大专项（2016ZX08003-001）

Digestive Stability of AM79 EPSPS Protein in Simulative Gastric and Intestinal Fluid

WANG Yu^1,2, WANG Jian-hua¹, LIU Yun-jun²

1. College of Agriculture,China Agricultural University,Beijing 100094;
2. Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081

Received:2017-02-10 Published:2017-06-26 Online:2017-06-19

摘要/Abstract

摘要： AM79 EPSPS基因是从草甘膦高度污染的土壤中克隆的、具有我国自主知识产权的一个新型抗草甘膦基因,在转基因玉米中过表达能显著提高转基因玉米的草甘膦抗性,具有重要应用价值。为了评价AM79 EPSPS蛋白的食用安全性,本研究参考中华人民共和国国家标准,开展了AM79 EPSPS蛋白的消化稳定性研究。将AM79 EPSPS基因构建到原核表达载体pET-30a上,在大肠杆菌中表达了分子量为48.3 kD的可溶性AM79 EPSPS蛋白。通过Ni-NTA亲和层析方法纯化了AM79 EPSPS蛋白用于体外模拟胃肠液实验。结果表明,AM79 EPSPS蛋白在模拟胃肠液中均在15 s内即被消化,SDS-PAGE和Western蛋白免疫印迹均未检测到蛋白残留,表明AM79 EPSPS蛋白在模拟胃肠液体系中极易被消化。

关键词: AM79 EPSPS蛋白, 草甘膦, 模拟胃肠液, 消化稳定性

Abstract: AM79 EPSPS is a novel glyphosate-resistant gene with independent intellectual property rights in China and isolated from highly glyphosate-polluted soil using metagenomics method. The gene owns a promising value because its overexpression in the transgenic corn increased the corns’ glyphosate resistances. In order to evaluate the safety of AM79 EPSPS protein as food,the digestive stability of AM79 EPSPS protein in simulative gastric and intestinal fluid was analyzed according to the national standard of the People’s Republic of China. Gene AM79 EPSPS was constructed into prokaryotic expression vector pET-30a,and the soluble AM79 EPSPS protein of 48.3 kD was expressed in Escherichia coli,and purified using Ni-NTA chromatography. The digestive stability of the purified AM79 EPSPS protein was performed via in vitro simulative gastric and intestinal fluid. The results showed that the AM79 EPSPS protein was completely digested within 15 s in the simulated gastric and intestinal fluid,no any remain of protein was detected by SDS-PAGE and Western blotting,indicating that AM79 EPSPS protein was easily digested in the simulated gastric and intestinal fluid.

Key words: AM79 EPSPS protein, glyphosate, simulative gastric and intestinal fluid, digestive stability

王玉, 王建华, 刘允军. AM79 EPSPS蛋白的体外模拟胃肠液消化稳定性研究[J]. 生物技术通报, 2017, 33(6): 176-181.

WANG Yu, WANG Jian-hua, LIU Yun-jun. Digestive Stability of AM79 EPSPS Protein in Simulative Gastric and Intestinal Fluid[J]. Biotechnology Bulletin, 2017, 33(6): 176-181.

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AM79 EPSPS蛋白的体外模拟胃肠液消化稳定性研究

Digestive Stability of AM79 EPSPS Protein in Simulative Gastric and Intestinal Fluid

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