Nguyen Hang Thu, Jehle Johannes A
Agricultural Service Center Palatinate (DLR Rheinpfalz), Department of Phytopathology, Laboratory for Biotechnological Crop Protection, Neustadt an der Weinstrasse, Germany.
Environ Biosafety Res. 2009 Apr-Jun;8(2):113-9. doi: 10.1051/ebr/2009015. Epub 2009 Oct 16.
The reliable use of purified Cry1Ab protein standards is a prerequisite for ecological studies and resistance monitoring programs of Cry1Ab-expressing transgenic corn. In this study the stability and activity of different Cry1Ab protein batches expressed in and purified from Escherichia coli were determined during two-year storage at different temperature conditions (4 degrees C, -20 degrees C, and -80 degrees C). SDS-Polyacrylamide gel electrophoresis showed degradation of the protein stored at 4 degrees C over four months, whereas no difference in the band intensity of the Cry1Ab proteins stored at -20 degrees C and -80 degrees C was observed. Bioassays with neonate larvae of Ostrinia nubilalis indicated that the biological activity of Cry1Ab varied from batch to batch, depending on the production process. Cry1Ab protein stored at 4 degrees C for four months showed a significantly decreasing activity measured as median lethal concentration (LC(50)), whereas the protein activity declined less than 11-fold after two years storage at -20 degrees C. When stored at -80 degrees C the toxin activity remained relatively stable for at least 30 months, as indicated by low LC(50) values of 7-10 ng Cry1Ab per cm(2) diet. These experiments demonstrate that appropriate long-term storage conditions of Cry1Ab protein standards are crucial for resistance monitoring programs of Bt corn, and storage at -80 degrees C is recommended.
可靠地使用纯化的Cry1Ab蛋白标准品是开展表达Cry1Ab的转基因玉米生态研究和抗性监测项目的前提条件。在本研究中，测定了在大肠杆菌中表达并纯化的不同批次Cry1Ab蛋白在不同温度条件（4℃、-20℃和-80℃）下储存两年期间的稳定性和活性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，在4℃储存四个月的蛋白发生了降解，而在-20℃和-80℃储存的Cry1Ab蛋白条带强度没有差异。用欧洲玉米螟初孵幼虫进行的生物测定表明，Cry1Ab的生物活性因批次而异，这取决于生产过程。在4℃储存四个月的Cry1Ab蛋白，以半数致死浓度（LC50）衡量，其活性显著下降，而在-20℃储存两年后，蛋白活性下降不到11倍。当储存在-80℃时，毒素活性至少在30个月内保持相对稳定，每平方厘米饲料中Cry1Ab的LC50值较低，为7-10纳克，表明了这一点。这些实验表明，Cry1Ab蛋白标准品合适的长期储存条件对于Bt玉米的抗性监测项目至关重要，建议在-80℃储存。
