Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera  

作　　者：Li-Zhen Chen Ge-Mei Liang Brian G. Rector Jie Zhang Kong-Ming Wu Yu-Yuan Guo 

机构地区：[1]State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China [2]USDA-ARS, European Biological Control Laboratory, Montpellier, France

出　　处：《Insect Science》2008年第6期497-503,共7页昆虫科学（英文版）

基　　金：This paper was contributed to the International Symposium on Insect Midgut Biology, April 7-11, 2008, Guangzhou, China.Acknowledgments This research was supported by the National Natural Science Foundation of China （30771424） and National Basic Research Program of China （973-2007CB 109204）. We thank Dr. Jun Zhao （University of West Virginia, USA） for reviewing this manuscript.

摘　　要：Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2- DE analysis.Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2- DE analysis.

关 键 词：Bacillus thuringiensis binding protein brush border membrane vesicles Cry1Ac Helicoverpa armigera two-dimensional electrophoresis 

分 类 号：Q965.9[生物学—昆虫学] Q51