Studies On Fermentative Technology Of E.coli Containing Eukaryotic Expression Plasmid With IBV Genes

The fermentation parameters of recombinant E.coli which contains the eukaryoticexpression plasmid pVAX1-S1, pVAX1-M, pVAX1-N, VR1020-S1, VR1020-M and VR1020-N were investigated.By a single factor experiment and orthogonal test, the fermentation parameters of recombinant E.coli which contains the eukaryotic expression plasmid with IBV genes in shake flask were investigated. In the experiment, the optimum medium was carried out and definited the medium ingredient. After fermentating, the optimum alkaline lysis was used to extract the plasmid DNA, and the yield could reach 27.8mg/L, also the purification was 1.88, which was 2.62-fold in comparison with using conventional LB culture.The fermentation parameters of recombinant E.coli which contains the eukaryotic expression plasmid with IBV genes in 5L fermentor were investigated. Inoculum size, temperature, pH and dissolved oxygen were optimized in the fermatator. pVAX1-M was definited to be the predominant strains. The optimum alkaline lysis was used to extract the plasmid DNA, and the yield could reach 57mg/L, also the purification was 1.88, which was 4.3-fold in comparison with using conventional LB culture.The fermentation broth was extracted by alkaline lysis and schizolysis. The alkaline lysis, such as schizolysis time and the ratio of the lysate were optimized. Then, PEG and Diatomaceous Earth were used to purificatd and compare. The extraction and purification technology was founded based on alkaline lysis and Diatomaceous Earth. This technology can move out foreign matter, such as most of the RNA, genome DNA and proteins. Meanwhile, the purification of the plasmid DNA can reach 1.88 and the ratio of superhelix DNA was 98.4%, which were consistent with the plasmid quality equirement of WHO. The thalli cultured in the fermentator were extracted and purificated by this technology, as a result, the yield of plasmid DNA can reach 57mg/L. The cost had cut down by 20-40% in comparison with traditional technology.This study cut down the production costs of fermentation medium and plasmid extraction. It provides an efficient and economical method for large-scale production of DNA vaccine.