Development and Study of Phage-Based Microarray and Dot-Blot
Type of DegreeThesis
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The sequencing of cell genomes along with the development of DNA microarrays established a foundation for the discovery of new genes and enabled the exploration of the whole transcriptome in a single assay . However, there is no absolute correlation between the mRNA transcription levels and the corresponding protein expression levels [2, 3, 4]. Hence, protein-specific high-throughput technologies are urgently needed. We developed a novel variant of a protein microarray, based on landscape bacteriophages where 4000 copies of specific peptides were displayed on the phage surface as an N-terminal part fused to the pVIII protein. In our model system, we used phages binding streptavidin and displaying the VPVGAYSDT or VPEGAFSS peptides. The control phage displayed the non - related EPRLSPHS peptide. All phages were immobilized on an epoxy - coated glass slide and the binding was monitored with fluorescently labeled streptavidin. We demonstrated that the sensitivity of our phage array is very high: VPVGAYSDT - phage specifically recognizes the target at concentration ~ 1.0 nM (60 fmoles in quantity). The system we described shows several advantages: 1) the robustness of the recombinant phage inherited from its natural predecessor (phage fd), which allows its use in harsh environments, 2) high density of the binding component to the total protein mass compared to antibodies and other proteins and 3) high sensitivity. The developed phage microarray was also adapted as a prospective high-throughput method for screening of phage clones specific for B. anthracis spores. An array variant fluorescent dot-blot was also designed for rapid examination of the discriminative selectivity of selected phage probes when exposed to mixed populations of spores of the Bacillus family.