Cry1Ie蛋白的模拟胃肠液消化稳定性及热稳定性分析

doi:10.13560/j.cnki.biotech.bull.1985.2015.11.028

摘要/Abstract

摘要： Cry1Ie蛋白对亚洲玉米螟(Ostrinia furnacalis)具有高毒力，cry1Ie基因已经应用于转基因抗虫玉米的种质创制。为评价Cry1Ie蛋白的食用安全性，开展了Cry1Ie蛋白的消化及热稳定性研究。利用实验室已经构建的表达载体，在大肠杆菌中表达了分子量为81 kD的可溶性Cry1Ie蛋白，经过Ni-NTA亲和层析和Superdex-75分子筛层析获得纯度达91%蛋白。模拟消化液实验结果表明，Cry1Ie蛋白在模拟胃肠液中15 s内即被消化，SDS-PAGE未检测到蛋白残留。热稳定性实验中，Cry1Ie蛋白在玉米粉提取液中不稳定，100℃ 30 min内蛋白基本降解。对样品进行了生物活性测定发现，经模拟胃肠液消化处理和热处理后的Cry1Ie蛋白对亚洲玉米螟无杀虫活性。Cry1Ie蛋白在胃肠液系统中和热处理条件下均不稳定，并丧失了对亚洲玉米螟的杀虫活性。

关键词: 转基因玉米, Cry1Ie蛋白, 消化稳定性, 热稳定性, 杀虫活性

Abstract: Due to the high virulence to Asian corn borer(Ostrinia furnacalis)of Cry1Ie, this protein had applied into the germplasm creating of transgenic insect-resistant maize. To evaluate the food safety of Cry1Ie protein, this study carried out the digestion and heat stability assays. Using the expressing vector which the laboratory has constructed, the Cry1Ie protein(81 kD)was expressed in Escherichia coli, and the high purity protein was obtained using Ni-NTA chromatography and Superdex-75 size-exclusion chromatography. The results of artificial digestion assay showed that Cry1Ie was degraded rapidly within 15 s in simulated gastric fluid and intestinal fluid, without any peptide left in SDS-PAGE. In the heat stability assay, Cry1Ie was not stable in the extracting solution of maize meal, and most was degraded within 30 min under 100℃. In the bioassay of the Asian corn borer(Ostrinia furnacalis), it suggested that Cry1Ie lost the bio-activity after the treatment of digestion and heat. In conclusion, Cry1Ie was not stable in the gastrointestinal fliud and the heat treatment, which had lost the bio-activity to the Asian corn borer(Ostrinia furnacalis).

Key words: transgenic, Cry protein, digestive stability, heat stability, insect-resistance

李欣竹, 耿丽丽, 高继国, 张杰. Cry1Ie蛋白的模拟胃肠液消化稳定性及热稳定性分析[J]. 生物技术通报, 2015, 31(11): 214-221.

Li Xinzhu, Geng Lili, Gao Jiguo, Zhang Jie. Analysis of Digestive Stability in Simulative Digestive Tract Fluid and Heat Stability of Cry1Ie Protein[J]. Biotechnology Bulletin, 2015, 31(11): 214-221.

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