- 學位論文
應用肝素化纖維素基質對未純化之第二型重組腺病毒溶液進行吸附與轉導
Application of heparinized cellulose matrices to adsorb unpurified rAAV2 from the crude lysate of packaging cells for binding and transduction
並列摘要
The microporous affinity membrane based on cellulose matrices offers minimal mass-transfer effects in membrane chromatography with low nonspecific adsorption. In this research, we tested a novel application of the microporous, heparinized cellulose matrices (H-CM) for their affinity toward recombinant adeno-associated virus serotype 2 (rAAV2, which uses heparan sulfate proteoglycans as the primary cellular receptor) to develop a controlled, substrate-mediated viral vector delivery. We adsorb rAAV2 from the crude lysate of packaging cells to an epoxy-crosslinked heparin cellulose membrane, which led to vector transduction upon cellular adhesion. When adhered, the human HT-1080 fibroblasts exhibited proliferation kinetics similar to those on the standard polystyrene tissue-culture surface. Using green fluorescent protein and beta-galactosidase as reporters, we showed that the heparin-bound rAAV2 particles remained active and that the rAAV2-heparin binding was reversible and capable of mediating transgene delivery in cell culture. This method avoids the use of conventional ultracentrifugation or chromatography in preparation of infectious rAAV2 for transduction. Our work explores a new application of affinity cellulose matrices in substrate-mediated viral vector delivery, which can be a useful tool in developing protocols for localized gene transfer.