WK蛋白在转基因抗旱小麦和大肠杆菌中的等同性验证

Equivalence verification of WK protein in genetically-modified drought-resistant wheat and Escherichia coli

目的作为转基因抗旱小麦安全性风险评价的重要前提,对转基因抗旱小麦中表达的蛋白和大肠杆菌外源表达蛋白进行等同性验证。方法本研究首先在大肠杆菌中表达了His-WK融合蛋白,然后提取了对照小麦种子和转基因抗旱小麦种子的总蛋白,最后通过SDS-PAGE凝胶电泳、蛋白免疫印迹、糖基化修饰检测和液相色谱-串联质谱(LC-MS-MS)技术分析WK蛋白在转基因抗旱小麦和大肠杆菌中的等同性。结果本研究在大肠杆菌中表达了His-WK融合蛋白,并成功进行了纯化;大肠杆菌中表达的His-WK融合蛋白和转基因抗旱小麦的WK蛋白的表观分子量和免疫原性具有一致性;大肠杆菌中表达的His-WK融合蛋白和转基因抗旱小麦的WK蛋白都没有翻译后糖基化修饰;大肠杆菌中表达的His-WK融合蛋白和转基因抗旱小麦的WK蛋白的一级结构具有一致性。结论本研究验证了WK蛋白在转基因抗旱小麦和大肠杆菌中的等同性,为后续转基因抗旱小麦在营养学、毒理学、致敏性、免疫性等安全性评价研究中提供了可靠的参考依据。

Objective As an important prerequisite for the safety risk assessment of genetically-modified drought-resistant wheat,it is to be verified in the present study that the equivalence of proteins expressed in genetically-modified drought-resistant wheat and exogenously expressed in E.coli.Methods At first,the His-WK fusion protein was expressed in E.coli,and then the total protein of control wheat seeds and genetically-modified drought-resistant wheat seeds was extracted;Finally,SDS-PAGE gel electrophoresis,Western blot,glycosylation modification and liquid chromatography-tandem mass spectrometry(LC-MS-MS)were performed to analyze the equivalence of WK protein expressed in genetically-modified drought-resistant wheat and E.coli.Results The His-WK fusion protein was expressed in E.coli and purified successfully;The apparent molecular weight and immunogenicity of the His-WK fusion protein expressed in E.coli and the WK protein of genetically-modified drought-resistant wheat were consistent;The His-WK fusion protein expressed in E.coli and the WK protein of genetically-modified drought-resistant wheat had no post-translational glycosylation modification;The primary structure of the His-WK fusion protein expressed in E.coli and the WK protein of genetically-modified drought-resistant wheat were consistent.Conclusion This study verifies the equivalence of WK protein in genetically-modified drought-resistant wheat and E.coli,and provides a reliable reference for the subsequent safety evaluation of genetically modified drought-resistant wheat in nutrition,toxicology,sensitization and immunity.