| Gene transfer to humans requires carriers for the plasmid DNA,which provide a efficient tool to carry the gene into the nucleus of the desired cells. A series of chemically different vectors are currently being investigated for these purposes. Major attention is paid to cationic polymers, which are able both to condense large genes into smaller structures and to mask the negative DNA charges. In this work, what we did is illustrated below: Firstly, Di-Fmoc-Lysine was prepared by Lysine and Fmoc-Osu and the effects of temperature,pH value and extracting method on yield and purity of the product were discussed in detail. The final product was characterized by HPLC and ESI-MS. It showed that α-and ε-amino groups of lysine could react with Fmoc-Osu at different pH value. Poly(lysine) dendrimer was synthesized using Di-Fmoc-Lysine as both branching agent and dendritic branch by solid phase peptide coupling method on Fmoc-Gly-Wang Resin. Poly(lysine) dendrimer was surface-functionalized by Fmoc-Phe and HAc. The product was characterized with 1H NMR and MALDI-TOF-MS. The results showed that products had exclusive molecular weights, indicating that could be developed to a potential ideal nonviral vector. For the great demand of polymers for plasmid, the enzyme-cutting analysis, transformation, assaying, extracting of production and the electrophoresis analysis of pGL3 plasmid have been studied in series. Finally we observed the condensation between the polymer and plasmid DNA by the methods of agarose electrophoresis, fluorescence spectrometer and Multi-angle laser light scattering (MALLS). The result demonstrated that a smaller polyplex with hydrodynamic radium of 32 nm could be obtained at a N/P ratio of 2. In this thesis, we have only done preliminary investigation about poly(lysine)/DNA polyplex, and we hope it can contribute to the further develop in this field.
|
PDF Full Text Request