Large-scale Production And Immune Application Of E.coli DH5?-sourced Avian Influenza DNA Vaccine

Avian influenza disease an emerging infectious disease caused by influenza A virus,which mainly affects the respiratory and nervous system of poultry.In China,avian influenza virus mainly focuses ontype of LPAI and HPAI,and several subtypes of virus co-infection areprevalent.With the outbreak of avian influenza in East,Middle and Western of China in recent years,thedatademonstrates more than 90% of avian influenza disease was caused by HPAI,which mortality attained to 70%,and lead to huge financial losses to the livestock breeding industry in China.Due to inoculationof inactivated vaccine is the best option for now,Agriculture Department follows the compulsory immunization measure to prevent and control HPAI.Because of highly mutation of avian influenza genes,HA genes of protective antigen particularly,H5 subtype of highly pathogenicavian influenza has produced several different antigen groups,however,vaccine of H5 avian influenza shows a time of relative lag,meantime,inactivated vaccine has the disadvantages of higher immune dose,stronger immune adverse effect and disturbing epidemic pathogen detection.Avian influenza DNA vaccine hasthe advantages of humoral immunity and cellular immunity motivation,less adverse effect and no virus spread,which offers an alternative option to inactivated vaccine.There has been commercialization of DNA vaccine in foreign country,and the first application for new drug certificate of the H5 DH5?-p CAGGopti HA5 DNA vaccine in China has already approved.Recombinant E.colicultivation and purification technique is widespread used in the laboratory for plasmid small scale preparation,however,when talked to large scale DNA vaccine production,there are several key points should be optimized to handle the difficult problems,including bacteria fermentation and high efficient purification.This study mainly focused on culture medium and fermentation condition optimization in 100 L and 1000 L fermenter,furthermore,efficacy of DNA vaccine and co-application was investigated to offer data for other products.Medium of DH5?-p CAGGopti HA5 DNA was optimized through the Response surface and Steepest ascent methods,the results analyzed by statistics showed that tryptone(10g/L),sucrose(17.5g/L),yeast(7.5g/L),ammonium sulfate(7.5g/L)and sodium chloride(10g/L)were the best medium ingredients.The DNA concentration was 115.25mg/L which produced using the 100 L fermenter,the yield increased by 88.52% in contrast to classical medium.The cultural character and genetic stability of the strain through serial passage was determined to screen the strain with high applicability.Fifteen strains from 15 passages were cryopreserved for further production,which verified with recombinant plasmid stability and purity.The culture condition including addition dose,dissolved oxygen and culture temperature was also determined for 1000 L fermenter cultivation.The results showed that the plasmid concentration attained to 359.2mg/Lwith the condition of 8% addition,30%dissolved oxygen and 5 hours inducing with 37.0 ? to42.0?,the yield increasedby 108.9%with the optimized medium in contrast to traditional culture medium,which provideddata for large-scale production.The DNA plasmid was extracted by modified alkalinelysis method and combined with lysis equipment,removing flocculefacility and tangential flow filtration.The result indicated that lysisefficiency improved by 15 times,plasmid recovery increased by18.74% with high purity.Efficacy of the DNA vaccine was detected by the animal experiment,the antibody titer of 5 weeks inoculated by 20?g dose could up to 7.3 log2.A large-dose of 200?g was inoculated to observe reaction for 14 days,and no systemic side effects were observed by the mental status and local reaction test.After two times of inoculation with 30?g dose,the antibody titer was higher than 4.5 log2,and the mean titer could up to 5.4 log2 for the 6 months trial.Through the study of the immune enhancement effect of avian influenza DNA vaccine and transfer factor combined immunization,the result indicated that the combined immunization of avian influenza DNA vaccine and transfer factor could improve the DNA vaccine immune efficacy by 1.2titer.The data provides reference data for the immune application of avian influenza DNA vaccine.