Large-Scale Production And Characterization Of Recombinant Plasmids

Gene therapy and gene immunization are hot research spots in current biomedical field. Among the gene transfer strategies under investigation, the non-viral gene transfer using the recombinant plasmids expressing the gene of interest has the overt advantages of good safety and ease of preparation over viral-mediated vectors. In terms of plasmid DNA preparation, currently-available commercial kits have the disadvantages of small-scale preparation, high cost and requirement of pretreatment using toxic chemicals, which are unsuitable for large-scale preparation of recombinant plasmids for animal use. As to other methods for large-scale production of recombinant plasmids such as anion-exchange chromatography, size-exclusion chromatography and hydrophobic chromatography, pretreatments using toxic chemicals such as phenol and chloroform and animal-derived RNase A are required, as well as time-consuming and high cost. To establish a suitable method for large-scale preparation of plasmid DNA for animal gene immunization/therapy, cost-effective culture media and the fermentation conditions were established for high-density fermentation of recombinant E.coli. Furthermore, the relationship between the plasmid replication and host bacterial growth was investigated in a 150-liter fermentator. The results showed that the growth state and the wet weight of recombinant DH5αand JM109 E.coli in the modified medium were better than that in standard LB medium. By shifting the culture temperature to 42℃after mid-log growth phase, the plasmid yield could be greatly improved.Other bottlenecks in the industrial production of plasmid DNA include disruption...