[Expression and analysis of biological activity of the recombination anthrax edema factor].
Anthrax toxin consists of three separate proteins, protective antigen (PA), lethal factor (LF) and edema factor (EF). EF is bacterial adenylate cyclase which, upon activation by its eukaryotic cofactor, calmodulin, causes a rapid increase in the intracellular cAMP level of host cells. EF can reduce the protective ability of host animal. In order to further research the mechanism of anthrax toxin, the expression plasmid was constructed and the structural gene for anthrax edema factor (EF) was expressed in Escherichia coli. Recombinant EF (rEF) was purified to homogeneity by a three-step procedure involving metal chelating affinity chromatography, cation-exchange chromatography and gel chromatography. From 1 liter of culture, 5mg of biologically active EF was easily purified. Rabbits were immuned with rEF, anti-EF antibodies were prepared and can neutralize rEF in cells. Tests in vitro proved rEF have good biological activity. rEF can competed the binding regions of PA with rLF in J774A.1 and CHO cells. rEF and rLF can restrain each other by competition. The successful expression of rEF has placed a solid foundation for the research on toxicity mechanism of EF, and screening for inhibitors against EF.
Dong
Da-yong
DY
Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China. dayong-dong@sohu.com
Xu
Jun-jie
JJ
Song
Xiao-hong
XH
Fu
Ling
L
Chen
Wei
W
D000262:Adenylyl Cyclases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology; D001427:Bacterial Toxins; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D009500:Neutralization Tests; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases