탄저 독소 검출 및 활성 저해를 위한 생체적합성 프로브 연구

Abstract

Anthrax is an acute infectious disease caused by Bacillus anthracis. In general, depending on infection types, the anthrax is classified as a cutaneous anthrax shown symptoms such as pruritus, furunculosis, and blister by skin contact of bacteria, and inhalation anthrax shown respiratory infection symptoms like a flu and pneumonia via breathe and mouth. All symptoms are caused by initiating that released toxins, Protective antigen (PA), Lethal Factor (LF), and Edema factor (EF) released from Bacillus anthracis, are interacted with anthrax toxin receptor (ATR) existed on cell surface. Among anthrax toxins, the LF is known to have vital role in mediating toxic effect as a Zn2+-dependent protease. Due to the released of high concentration of anthrax toxin led to acute pathogenicity, thus the anthrax toxins have been remain an attractive target for the design of novel inhibitors that can efficiently neutralize the toxin and also used as a diagnosis index to detect bacterial infection. Similarly, PA have also been majorly focused as a theragnostic target since PA binds to ATR existed on host cell followed by endocytosis complex leading to toxicity. In this research, there developed a highly specific peptide and RNA aptamer as diagnostic and inhibitory probe against PA, ATR, and LF. Further, in order to improve the sensitivity and inhibition potency of the identified probes, a novel multivalent probe systems are developed. The probe binding efficiency were measured by immunocytochemistry (ICC) test and fluorescence activated cell sorting (FACS) analysis, and the inhibition potency for each probes was measured by in vitro proteolytic assay on Native-PAGE gel and cytotoxicity test on Raw 264.7 macrophage cell. In the study of diagnostic system for anthrax toxin, two different type detection systems using PDPP and aptamers binding to PA on nanostructures were developed. The first detection system is applied on nonporous ZnO which roles enhancing fluorescence signal with PDPP, and another one is applied on reduced graphene oxide-field effect transistor (RGO-FET) with aptamers binding to PA63. Taken together, based on the results in this study, it suggest that the biocompatible small molecules such as peptide and aptamer and polyvalent peptide were effective probes for detection and inhibition of anthrax toxin in anthrax disease. It is further suggested that the conjugation of identified probes with the various nanomaterials would enhance their physicochemical stability and thus lead to the development of early and sensitive diagnostic system for anthrax disease. Briefly, the development of difunctional (diagnosis and inhibition) biocompatible small probes or multivalent probes could be effectively used for other disease diagnosis and therapy.