Investigation of conjugation reaction synthetic peptides of Influenza A virus with polyelectrolytes by fluorescence resonance energy transfer and the other methods

Influenza A virus had caused epidemics and pandemics all over the world at different times and probably will cause in the future, too. Lastly in 2009, WHO raised the level of pandemic alert from phase 5 to 6 because of colloquially known as Swine Flu caused by Influenza A Virus. In this PhD. thesis, four different peptide sequences of the influenza A virus was used. Two of them were synthesized in our laboratory and the other two sequences were purchased commercially. These peptide sequences were characterized. Four different carriers (polyacrylic acid, poly(N-vinyl-2-pyrrolidone-co-acrylic acid), dextran aldehyde, and bovine serum albumin) were used for the synthesis of the peptide polyelectrolyte bioconjugates and formation of the complexes. Polyacrylic acid and bovine serum albumin were purchased commercially. Poly(N-vinyl-2-pyrrolidone-co-acrylic acid) and dextran aldehyde were synthesized and characterized in our laboratory. Bioconjugates and complexes of different peptide epitopes of Influenza A Virus were synthesized through the mentioned carriers with various methods. Physicochemical structures of these bioconjugates and complexes were investigated, in order to increase immunogenity of the peptide antigens, the necessary three-dimensional structures of peptide bioconjugates were formed and characterized. The formation of bioconjugates and complexes were clarified by various spectroscopic and chromatographic methods. When results were compared, bioconjugates formed with xxviiicovalent bonds which were obtained in the presence of carbodiimide was found to be more efficient.For the Influenza A virus M2 peptide polyelectrolyte bioconjugates designed as a synthetic vaccine model; it didn?t show significant toxic effect on the MCF-7 cells using low concentrations in vivo applications; by controlling cell viability with Flow Cytometry method and MTT experiments, results that supporting each other have been obtained and the usability of bioconjugates upon the experimental animals have been presented.The formation of conjugation reaction was investigated by Fluorescence Resonance Energy Transfer method with using Fluorescence dye. By calculating intermolecular distance, conformation changes in the pure peptide and the formation of bioconjugate were calculated and expressed with numerical values. In the literature, the peptide sequence of Influenza A virus Hemagglutinin H3N2 91-106 was modeled for the first time, the optimized geometry with minimum energy were determined by molecular mechanics method. The numerical values obtained that are based on experimental data proved to overlap the theoretically calculated values by FRET method. In this PhD. thesis, with the information obtained from intermolecular distance calculations between donor and acceptor in bioconjugate by Fluorescence Energy Transfer Method clearly showed to obtaining of specific antibodies against the disease because of the epidemics and pandemics caused by Influenza A viruses, the creation of nanobiosensors and diagnostic kits and the most important; target of our teacher, precious scientist, decedent Prof. Dr. Mehmet Mustafaev Akdeste, the development of multifunctional biotechnology vaccines with binding of more than one disease's antigen on the same polymer matrix.Keywords: Influenza A virus, Fluorescence Resonance Energy Transfer, Covalent Conjugation, Optimized geometry, 3-dimensional conformation