The Establishment Of Novel Fluorescence Resonance Energy Transfer Systems And Their Applications In The Detections Of Nucleic Acid And Antibody

Kras gene, one of the oncogens, is frequently mutated in tumors. It works like a "switch" of organism that regulates cell growth and tumor angiogenesis in the process of signal transduction, effecting the growth and diffusion of tumors. The detection of Kras gene could provide an important basis for clinical tumor diagnosis, patient condition valuation and prognosic judgment, and gene therapy. Due to the role of Kras gene in tumor diagnosis and therapy, it has necessitated the development of a quantitative detection method for the mutated Kras gene.As a type of interleukin, interleukin-8 (IL-8) is a pleiotropic and overlapping factor with biological activity. It has the contribution to regulate the differentiation and maturation of hemocytes, dominate the immune response and relate with a series of cancers. Nowadays humanized anti-IL-8 monoclonal antibody has been used in clinical studies on related diseases. As a result, design a fast and efficient detection of anti-IL-8 monoclonal antibody would supply favorable conditions for inflammation and cancer research.In this thesis, different fluorescence resonance energy transfer (FRET) systems have been systematic studied. In addition, we have successfully applied these FRET systems to nucleic detection and immune analysis, including quantification of mutated Kras gene and biotinylated anti-IL-8 monoclonal antibody.The first chapter is an introduction part to summarize the FRET principle, theorical calculation methods, researches of fluorescent probes and the FRET applications of biological analysis. The aspects of nucleic acid detection, immunoassay and protein-protein interaction have been described in detail.In the second chapter, the FRET realized by combination with surfactant CPB has been detailed studied. It is found that cationic surfactant could be used to enhance the FRET signal by its compression effect on DNA strands. The FRET between donor and acceptor with large spacing has been achieved in a separation-free format and the quantification of target DNA of 30 bps has been completed. In addition, to study the interaction of CPB and double-stranded DNA, CD spectra have been utilized and then the experimental phenomena have been reasonably interpreted.In the third chapter, the FRET sensitized by combination with surfactant CTAB has been studied in detail. In this system, FRET is realized by the interaction between cationic surfactant and quantum dot with the secondary antibody labled with dyes. It is illustrated that antibody does not need to be marked on specific regions or processed to special fragments. Based on the increase of acceptor fluorescence peak area caused by FRET, quantitative analysis of biotinylated IL-8 antibody has been achieved.In the fourth chapter, the FRET system used DNA intercalating chromophore JOJO-1 as donor has been studied. It is found that using JOJO-1 as donor to form FRET system, the capture DNA does not need to be labeled, the false positive could be effectively decreased and the selectivity could be improved. Based on the increase of acceptor fluorescence peak area caused by FRET, target DNA containing 30 bps has been successfully quantified.The main characteristics of this thesis are as follows:1. The effects of surfactants on FRET systems have been studied and two novel quantification approaches of long-stranded nucleic acids and antibody have been established by utilizing their interaction.2. The FRET system used DNA intercalating chromophore JOJO-1 as donor has been studied and the approach for nucleic acids detection has been established, which has a high signal to nosie ratio and high selectivity.3. The above researches provide a certain theoretical and experimental basis to explore novel FRET systems and select novel fluorescence probes, which have potential value of applications to the analysis of nucleic acids, antibody and other macromolecules.