Effective Production And Ex Vivo Evaluation Of Plasmid Vector Intended For Human Cancer Gene Therapy

Effective Production and Ex vivo Evaluation of Plasmid Vector Intended for Human Cancer Gene TherapyABSTRCTThe effective production of plasmid vector intended for gene therapy was investigated systematically. Plackett-Burman method was employed to determine the most important components in the MBL medium with regard to plasmid production and cell growth. The effects of these components on plasmid yield, cell mass and specific plasmid DNA productivity were further evaluated in shake-flask scale, and an optimal MMBL medium was formulated. The results showed that glucose was an optimal carbon source with the optimal concentration of 5.0g/l, and glycerol could be chosen as a complementary carbon source. The optimal nitrogen source concentration (yeast extracts 15g/l, casamino acids 20g/l) was also determined. Further study showed that the initial acetate addition (less than 2.0g/l) in MBL would improve plasmid production significantly, although cell growth was inhibited. The effects of temperature and antibiotics on plasmid production were also studied, with the optimal temperature as 36 癈. Batch and fed-batch fermentation were performed on 21 bench-top fermentor, and 75mg/l plasmid production obtained. The initially purified plasmid was used to transfect the gastrointestinal tumor cell ex vivo, and apoptosis phenomena was observed. The cloning vector of apoptin, derived from chicken anemia virus, was constructed successfully, and the expression vector was constructed in progress.