EPSPS蛋白和PAT蛋白的模拟胃液消化稳定性分析

Digestive stability in simulated gastric fluid of EPSPS protein and PAT protein

目的研究5-烯醇式丙酮酸莽草酸-3-磷酸合成酶(5-enolpyruvylshikimate-3-phosphate synthase,EPSPS)蛋白和膦丝菌素乙酰转移酶(phosphinothricina cetyltransferase,PAT)蛋白在模拟胃液中的消化稳定性。方法参照中华人民共和国国家标准(农业部869号公告-2-2007)模拟胃肠液外源蛋白质消化稳定性试验方法中模拟胃液的配方,在体外建立模拟胃液消化体系,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(dodecyl sulfate,sodium salt-polyacrylamide gel electrophoresis,SDS-PAGE)和蛋白印迹(Western blot),分析EPSPS蛋白和PAT蛋白在模拟胃液中不同消化时间点的降解情况。结果EPSPS蛋白和PAT蛋白均在15 s内消化完全,SDS-PAGE和Western blot蛋白免疫印迹均未检测到蛋白残留,EPSPS蛋白和PAT蛋白在模拟胃液中极易被消化。结论EPSPS蛋白和PAT蛋白经过胃液消化后,不具有免疫原性。

OBJECTIVE To study the digestive stability of 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)protein and phosphinothricina cetyltransferase(PAT)protein in simulated gastric fluid.METHODS The component of simulated gastric fluid was based on the method of target protein digestive stability in simulative gastric and intestinal in national standard of the People’s Republic of China(Published by the Ministry of Agriculture No.869-2-2007).The test model of stability of different protein to digestion in Simulated Gastric Fluid was established by dodecyl sulfate,sodium salt-polyacrylamide gel electrophoresis(SDS-PAGE)and western blot.The degradation of EPSPS protein and PAT protein in simulated gastric fluid at different digestion time points were analyzed.RESULTS The experiment showed that EPSPS protein and PAT protein were completely digested within 15 s in simulated gastric fluid,no any remain of protein was detected by SDS-PAGE and Western blot,indicating that EPSPS protein and PAT protein were easily digested in the simulated gastric.CONCLUSION EPSPS protein and PAT protein do not have immunogenicity after digestion with simulated gastric fluid.