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目的评价转Cry1Ab/Cry2Aj、G10evo(EPSPS)基因抗虫耐草甘膦玉米“双抗12-5”中的外源基因重组蛋白在模拟胃肠液中的消化稳定性。方法根据国家标准建立体外模拟胃肠环境消化体系,样品或对照都以5 mg/ml的浓度模拟胃液消化,2 mg/ml的浓度模拟肠液消化。分别于反应0 s、15 s、2 min、30 min和60 min时取出200μl模拟消化液,加入上样缓冲液并沸水浴5 min终止反应,再进行十二烷基磺酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)、染色并观察结果。结果大豆胰蛋白酶抑制剂(soybean trypsin inhibitor,STI)在模拟胃、肠液中60 min仍不能被全部消化,酪蛋白(α-casein)在模拟胃、肠液中0~15 s内全部消化,Cry1Ab/Cry2Aj重组蛋白在模拟胃、肠液中0~15 s内完全消化,G10evo(EPSPS)重组蛋白在模拟胃液中0~15 s内全部消化,在模拟肠液中2~30 min内全部消化。结论转Cry1Ab/Cry2Aj、G10evo(EPSPS)基因玉米的外源基因重组蛋白在模拟胃、肠液中不具有消化稳定性,容易被降解。
Objective To evaluate the digestive stability of exogenous gene recombinant proteins in Cry1Ab / Cry2Aj, G10evo (EPSPS) -resistant insect-resistant glyphosate-tolerant maize “double-resistant 12-5 ” in simulated gastrointestinal fluid. Methods According to the national standard, a gastrointestinal environment digestion system was established in vitro. All the samples or controls were simulated gastric juice digestion at a concentration of 5 mg / ml and simulated intestinal fluid digestion at a concentration of 2 mg / ml. 200 μl of simulated digestion solution was taken out at 0 s, 15 s, 2 min, 30 min and 60 min respectively, and the reaction was stopped by adding a loading buffer and a boiling water bath for 5 min. Sodium dodecyl sulfate-polyacrylamide Gel electrophoresis (SDS-PAGE), stained and observed the results. Results Soybean trypsin inhibitor (STI) could not be digested completely in simulated gastric and intestinal fluid for 60 min. Casein (α-casein) was completely digested in simulated gastric and intestinal fluid within 0 ~ 15 s. Cry1Ab / The Cry2Aj recombinant protein was completely digested within 0-15 s in the simulated gastric and intestinal fluids. The G10evo (EPSPS) recombinant protein was completely digested within 0-15 s in simulated gastric fluid and completely digested in simulated intestinal fluid within 2-30 min. Conclusion The exogenous gene recombinant proteins of transgenic Cry1Ab / Cry2Aj and G10evo (EPSPS) maize do not have digestive stability and are easily degraded in simulated gastric and intestinal fluid.