Study On Preparative Protocol Of PCD-Awte Candidate Malaria DNA Vaccine

DNA vaccine,which could induce cellular and humoral immunity, is one of the most promising candidate vaccine for prevention, treatment and cure of diseases such as AIDS and malaria. There is an increasing interest and need for the development of scaleable process for the production of pharmaceutical-grade plasmid DNA for DNA vaccine. However,methods that were designed to produce small amounts of plasmid DNA for laboratory use can not be adapted to produce pharmacological quality and quantities. It is necessary to establish a suitable preparative protocol with which the vaccine can be applied in clinical research as soon as possible.The pCD-Awte candidate malaria DNA vaccine has been shown to be highly effective when using mice and orhesus monkeys as evaluation model, a standard and controllable plasmid DNA preparation protocol is therefore in urgent need. Here we established an efficient plasmid DNA production method with high yield and purity. Firstly, the engineered bacteria pCD-Awte/DH5αwas constructed in order to enhance plasmid stability in fermentation culture, which will be dependent on the interactions between the host organism, the recombinant plasmid vector and the growth environment. Secondly, Escherichia coli DH5αcontaining recombinant plasmid pCD-Awte was cultured. Several parameters related to fermentation have been tested, including different nutrient constitute of medium, different feed medium, and temperature conversion to investigate the effects of plasmid DNA production through fermentation culture of shaking flask and 5L fermentor. The result showed that the Mg²⁺, trace element solution, foreign nucleosides, temperature conversion from 37℃to 42℃and glucose instead of glycerol as feed-medium can increase the yield of plasmid DNA, the yields up to 122-130 mg in 1L culture solution was obtained in the optimization conditions.After fermentation, crude plasmid DNA was released from Escherichia coli DH5αby alkaline lysis. With subsequent clarification and concentration, the supernatant was further purified with a four-step efficient chromatographic procedure to eliminate host cellular RNA, proteins, genomic DNA, endotoxins and plasmid DNA isoforms. The procedure included Sepharose6FF size exclusion chromatography, Plasmidselect thiophilic aromatic chromatography, Source 30Q anion exchange chromatography and G25 size exclusion chromatography. Several parameters related to chromatographic procedure have been optimized to this vaccine, including sample volume, flow-rate and colunm volume. Lastly, the purified plasmid DNA was identified, the host cellular RNA and plasmid DNA isoforms were identified by agarose gel electrophoresis, the host cellular proteins was assayed by ELISA, the genomic DNA was measured by real-time PCR and the endotoxins was identified by TAL. The result showed the quality of purified plasmid DNA met the standards of pharmaceutical-grade plasmid DNA.