Study On Tissues Distribution And Preparation Technology Of Avian Influenza DNA Vaccine

For medium sized test and drug application, several studies including tissues distribution and preparation technology of plasmid DNA were performed on the basis of Avian Influenza DNA vaccine pCAGGoptiH5. 21-day-old SPF chickens were intramuscularly immunized with dose of 10ug Avian Influenza DNA vaccine pCAGGoptiH5. Blood, heart, liver, spllen, lung, kidney, stomach, intestines, brain, injecting site muscle of immunized SPF chickens was detected by PCR method on 1h,3h,1d,3d,7d,15d,30d,50d,80d,120d,150d,200d,280d after injected. Results revealed that plasmid DNA existed in all objective tissues one hour after immunized, existed in blood 15 days, existed in injecting site muscle 280 days after immunized. Furthermore, prevenient experimental datas proved Avain Influenza DNA vaccine pCAGGoptiH5 could offer complete protection to SPF chickens. To satisfy the demands of medium sized test, we have studied the preparation technology of DNA vaccine. The preparation technology of plasmid DNA which using fed-bath culture and temperture-changing technology to culture recombinant E.coli in fermentor,and to lysis cells by traditional alkaline-based plasmid preparation method, and to filtrate plasmid DNA by hollow fiber ultrafilter, and to purify plasmid DNA by anion exchange chromatogram. Relatively on the basis of no criterion, we have established quality criterion to identify purified plasmid DNA on four aspects: purity , digestion analysis , PCR, immunocompetence. The results showed that the purified plasmid by the preparation technology was not only stable, but also was high immunocompetence.