RESUMO
Specific identification of Bacillus anthracis (B. anthracis) is vital for the accurate treatment of afflicted personnel during biological warfare situations and civilian terrorist attacks. In order to accomplish this, we have subjected the lysates from B. anthracis to affinity purification using monoclonal antibodies for the selected antigenic protein present in the bacteria. The bound antigenic protein was identified by multi-dimensional protein identification technology (MudPIT) to be a surface layer protein EA1. The same antigen was identified from the lysates from a few strains of B. anthracis demonstrating the observation to be common for B. anthracis strains. Hence, this presents an effective pathway for the identification of the bacteria present in unknown samples of various origins. Generation of a database containing the EA1 protein has been found to be useful in the database search of unknown samples.
Assuntos
Bacillus anthracis/química , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos , Anticorpos Monoclonais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/classificação , Bacillus anthracis/genética , Bacillus anthracis/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Guerra Biológica , Bioterrorismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Espectrometria de Massas em Tandem/métodosRESUMO
We report herein a new method for the aminoacylation of tRNA, using a resin-immobilized ribozyme and the cyanomethyl ester (CME) of an amino acid substrate. The oxidized form of the ribozyme was immobilized on a hydrazine resin via covalent linkage. We performed aminoacylation of tRNAs using this ribozyme-resin to isolate aminoacyl-tRNAs. The column was recycled up to 5 times without significant activity loss. Thus, our ribozyme-based aminoacylation system has significant potential to be a powerful and practical technique for supplying various nonnatural aminoacyl-tRNAs for a highly efficient in vitro translation system.
