Research On The Fermentation And Purification Of HIV-1 DNA Vaccin

AIDS caused by HIV is one of the most serious threaten to human health. Owing to the unique infection mode, the tradition vaccine could not build transparent protection. DNA vaccine, which could induce cellular immune and humoral immune responses as well as long term memorial immune response, is one of the most hopeful HIV vaccines, which lead to the attention of he medium prepare of clinical plasmid DNA. The DNA vaccine carrier pDRVI2.0K and non-resistant DNA vaccine of HIV-1 CN54 which is the main epidemic strain in China has been built.In the flask, the carbon, nitrogen source and inorganic salt is optimized through orthogonal experiment, which adding to the M9 midia. High density culture of DH5 a /pDRVI2.0Kwas achieved using the optimized semi-synthesized medium in the 5Lfermenter. Also the incubate condition including temperature, pH, feeding time and feeding mode are optimized. The OD600 value of the culture of DH5/pDRVI2.0K reached 39, and the plasmid yield reached 90mg/L after 20-hours incubating in the fed-batch culture in the optimized condition.And DH5/pDRVI2.0-gpnef which was cultivated under no selection pressure, the yield and stability of plasmid in batch culture and fed-batch culture were compared. The result indicated, fed-batch culture could lead to the increase of thallus but without the increase of plasmid concentration.Plasmid purity reached standards for clinical use after a process of alkaline lysis, ion-exchange chromatography, gel filtration and selective precipitation.At last the research and mass prepare of DH5 a /pDRVI2.0K-gpnef and DH5 a /pDRVI2.0K-gpl40 are proceeded under GMP condition.