In Vitro Digestion of Cry1Ab Proteins and Analysis of the Impact on Their Immunoreactivity
In Vitro Digestion of Cry1Ab Proteins and Analysis of the Impact on Their Immunoreactivity
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DOI：
10.1021/jf903189j
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发表时间：
2010-03-10
影响因子：
6.1
通讯作者：
Adel-Patient, Karine
中科院分区：
文献类型：
--
作者：
Guimaraes, Valeria;Drumare, Marie-Francoise;Adel-Patient, Karine
在pH 1.2和胃蛋白酶与蛋白质的高比例下进行的胃蛋白酶抗性试验是用于评估新蛋白质变应原性的证据权重方法的步骤之一。然而，已经提出了使用其他体外消化率试验，在更生理相关的条件下进行，并与免疫学测定相结合，以增加从新蛋白质消化稳定性研究中获得的信息的价值，用于整体变应原性评估。本研究的目的是研究Cry 1Ab原毒素和毒素，在转基因作物中表达的杀虫蛋白，使用模拟胃液（SGF）在不同的pH值和胃蛋白酶-底物比，在存在或不存在生理表面活性剂磷脂酰胆碱（PC）的消化的稳定性。电泳和免疫印迹模式和残留的免疫反应性进行了分析。尽管Cry 1Ab原毒素在pH 1.2下以高胃蛋白酶与蛋白质比率广泛降解，但其在pH 2.0下仅轻微降解并保留其免疫反应性。此外，Cry 1Ab蛋白在生理学相关性更高的体外消化率试验（pH 2.5，胃蛋白酶与底物的比例为1：20（w/w），含PC）中表现出稳定性。诸如pH、SGF组成和胃蛋白酶与底物的比例等因素则极大地影响Cry 1Ab蛋白的消化，从而证实还应考虑新的和生理学上更相关的体外消化率测试来研究蛋白质对消化的抗性与其过敏性之间的关系。
A pepsin resistance test performed at pH 1.2 and with high pepsin to protein ratio is one of the steps of the weight-of-evidence approach used for assessment of allergenicity of new proteins. However, the use of other in vitro digestibility tests, performed in more physiologically relevant conditions and in combination with immunological assays so as to increase the value of the information gained from the studies of stability of a novel protein to digestion for the overall allergenicity assessment, has been proposed. This study then aimed to investigate the stability to digestion of Cry1Ab protoxin and toxin, insecticidal proteins expressed in genetically modified crops, using simulated gastric fluid (SGF) at different pH values and pepsin-to-substrate ratios, in the presence or absence of physiological surfactant phosphatidylcholine (PC). Electrophoresis and immunoblot patterns and residual immunoreactivity of digesta were analyzed. Although Cry1Ab protoxin is extensively degraded at pH 1.2 with high pepsin-to-protein ratio, it is only slightly degraded at pH 2.0 and conserved its immunoreactivity. Furthermore, Cry1Ab proteins were demonstrated to be stable in a more physiologically relevant in vitro digestibility test (pH 2.5, pepsin-to-substrate ratio 1:20 (w/w) with PC). Factors such as pH, SGF composition, and pepsin-to-substrate ratio then greatly influence the digestion of Cry1Ab proteins, confirming that new and more physiologically relevant in vitro digestibility tests should be also considered to study the relationship between the resistance of a protein to digestion and its allergenicity.