| This subject was based on the study on the field experiment and biosafety of bovine tuberculosis trivalent DNA vaccine,we study on semi-works production technique to the engineering bacteria, which contian the trivalent DNA,and purificated the DNA vaccine in a bench scale. Finally we evaluated the immune effect preliminary by immunized animal bovine and mice.Firstly,E.coli engineering bacteria(DH5a), which contained recombinant eukaryotic plasmids pjw4303-mpt83, pjw4303-mpt64, pjw4303-Ag85b, were subjected to pilot-scale fermentation study under laboratory conditions. three different culture media were screened in a small scale using shake-flasks, semi-synthetic medium exhibited high density fermentive potential to selected as the pilot-scale medium for fermentation tank; and plasmid stability test aimed at this medium was applyed. Type NBS110 14L fermentation tank was used, the corresponding conditions were as follows:batching culture, the optimal mode of fed-batch was gradient feeding at a constant rate,temperature in the range of 37-42℃, dO2 at approximately 45%, pH at 7.0, rotational speed controlled at 200-600rpm. the results showed that bacteria density(A260/280), bacteria wet mass, plasmid yield was 22.92,27.173g/L,1.44mg/g , respectively. This technique has greatly saved the cost of vaccine production, laid a foundation for the production of bovine tubercle bacillus DNA vaccine, and was applicable to the large-scale culture of engineering bacteria.Secondly.on the base of high density fermentive engineering bacteria, plasmids was isolated rudely through conventional alkaline lysis. Major RNA in the alkaline lysate was removed by calcium chloridize, and clarified solution was obtained by Sartobran P bladder-type filter; plasmid super-coil DNA was precipitated, and major bacteria proteins and host genomic DNA were removed by polyethyene glycol method.Finally,endotoxin value before and after loading into source 30Q anion exchange column was 0.09-0.15 and<0.0197, respectively.It was suggesting that anion exchange column could significantly reduce endotoxin content. A26o/A2so of purified DNA was 1.84-1.88, proteins were not detected, suggesting that the DNA vaccine purified by this technique could meet the requirements of animal immune tests. This technique achieved one-step chromatographic purification of DNA vaccine, with the advantages of simplicity and rapidity.Thirdly,to examine the immune effect of purified vaccine, immune effect evaluation was operated on mice and bovines, respectively. In the examination of antibody level in mice humoral immunity, the antibody level of partial mice reached 1:128003 weeks after third immunization, relative to 1:200 after first immunization, suggesting that the DNA vaccine could strongly trigger the humoral immune response in mice. In the examination of IFN-y in mice cell immunity, the content of specific IFN-y was: Ag85B,234.19±69.8; MPT64,115.2±55.38; MPT38,179.61±66.26,7-15 times than the group of PBS and empty vector. The results examined in bovine humoarl immunity antibody level indicated:the level reached 1:32003 months after third immunization, suggesting that the DNA vaccine can induce the cattle and mice's specificity immunological reaction,however, the comprehensive immune effect need the data of conteracting toxic substances in the feature days.but the immune effect were not diversity after use the DNA vaccine which purificed with the commercial kit and our preparation technology.which suggested that our precipitated vaccine did not declined the immune effect,this can be declarate our preparation technology advisable and effective.
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