Gene Therapy Using Purified Plasmid Dna Production And Product Research

A large number of domestic and international studies showed that the IL-24 gene has a broad-spectrum anti-tumor activity, which can inhibit the melanoma, cervical cancer, lung cancer, prostate cancer tumor cells. Plasmid which contains IL-24 gene has potential application prospect. Large-scale preparation and purification of pharmaceutical grade plasmid DNA which contains IL-24 gene are the key for gene therapy. The thesis is composed of five sections:the construction of engineering strain which contains the recombinant plasmid pcDNA3.1-IL-24, fermentation,large-scaled alkali lysis conditions; the isolation and purification of recombinant plasmid pcDNA3.1-IL-24; the establishment of quality inspection system.First of all, rh-IL-24 engineering strain was set up by transforing recombinant plasmid pcDNA3.1-IL-24 into E.coli DH5a. When the stability of engineering strain was investigated, the fed-batch fermentation system was set up in the BIOFLO 40L fermentor. The fermentation parameters were as follows:fermentation temperature was 36℃～38℃, the dissolved oxygen concentration was controlled between 30%～50%, pH was about 7.0. The yield of E.coli cells had reached 36g/L after 16 hours fermentation. Bacterias were collected by centrifugation. After alkaline lysis, the plasmid DNA was roughly purified through the salt precipitation, and then further purified by the ion-exchange chromatography, hydrophobic chromatography and molecular sieve chromatography. At last, the plasmid DNA was loaded into the ampoule after ultrafiltration.Corresponding detection system was set up according to "Pre-clinical Studies Technical guiding principle based on Prevention with the DNA vaccine ".The result showed that:there was not impurity in the plasmid DNA; pH was 7.2; concentration was 727μg/mL; purity (OD260/OD280) was 1.87; the concentration of impurity protein was 0.007μg/μg plasmid DNA; there was not RNA and antibiotic residues; the concentration of endotoxin was less than 0.007EU/μg plasmid DNA. The results met the medical standards. Compared with the traditional laboratory methods, the process avoided the use of toxic reagents (phenol, chloroform) and animal-derived enzymes (RNase A), so it had not only ensured the safety of the product, but also reduced the complicated detection steps. It was money-saving, the price of agents were cheap and the materials and equipments (gel, reactor, etc.) can be repeatedly used. The procedure also sufficed the production with a short cycle production. In addition, it established the corresponding quality testing system for a large-scale pharmaceutical grade plasmid DNA preparation which of important significance. The study provided an efficient and economical method for large-scale production of therapeutic plasmid DNA vaccine, it had significant practical importance for the industrialization production of the DNA vaccine, it had offered the theoretical foundation to similar studies.