Method for screening inhibitors of the toxicity of Bacillus anthracis

Patent · Mon Jan 01 00:00:00 EST 2001

OSTI ID:874161

Cirino, Nick M ^[1]; Jackson, Paul J ^[1]; Lehnert, Bruce E ^[1]

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1. Los Alamos, NM

The protective antigen (PA) of Bacillus anthracis is integral to the mechanism of anthrax poisoning. The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has also been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins were capable of binding to specific cell surface receptors as determined by fluorescent microscopy and a flow cytometric assay. To confirm binding specificity in the flow cytometric assay, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. This assay can be employed as a rapid screen for compounds which disrupts binding of PA to cells. Additionally, the high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning.

• Research Organization: Univ. of California (United States); Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
• Sponsoring Organization: USDOE
• DOE Contract Number: W-7405-ENG-36
• Assignee: The Regents of the University of California (Los Alamos, NM)
• Patent Number(s): US 6329156
• Application Number: 09/273,839
• OSTI ID: 874161
• Country of Publication: United States
• Language: English

Related Subjects

method
screening
inhibitors
toxicity
bacillus
anthracis
protective
antigen
integral
mechanism
anthrax
poisoning
cloning
expression
purification
32
kda
fragment
pa32
described
expressed
fusion
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stabilized
green
fluorescent
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egfp-pa32
proteins
capable
binding
specific
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surface
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determined
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flow
cytometric
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confirm
specificity
non-fluorescent
pa83
competitively
inhibit
employed
rapid
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compounds
disrupts
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additionally
intracellular
levels
ease
recombinant
attractive
vaccine
candidate
therapeutic
treatment
/435/436/