绿脓杆菌PA—103株产毒条件及外毒素A提取纯化的研究

Study on Toxin-secreting Condition of Pseudomonas Aeruginosa PA-103 and the Extraction and Purification Exotoxin A

绿脓杆菌PA—103株产毒培养基是用本室研制的胰蛋白酶消化大豆粉透析外液制成的.培养基除铁后Fe^+含量为0.13μm/ml,蛋白水解物6.9mg/ml,氨基酸含量76mg/100ml,均接近进口培养基水平.经振荡培养,提取纯化了外毒素A.粗制外毒素A经DE_(52)阶段洗脱,DE_(52)梯度洗脱,LKBACA_(44)超凝胶过虑以及羟基磷灰石柱等四步纯化.精制外毒素A小鼠毒性致死试验提高到1:64,接近文献值.LD50>300LD50/ml,园盘电泳分析为单一成份,由天门冬氨酸等17种氨基酸组成.

The toxin-secreting culture medium of pseudomonas aeruginosa PA-103 is made in our laboratory, which is the outer dialysis solution of soybean powder digested by trypticase. The Fe++ content is O.13μg/ml after removing Fe + + , hydrolysated protein content is 6.9mg/ml, Amino acidcontent is 76mg/100mi, all approach the standards of imported medium. Affter shaking culture, extract the crude exotoxin A is purified by DE52 phase clution, DE52 linear gradient clution, LKB AcA44 ultrogel filtration and hydroxylapatite column. The refined exotoxin A toxicity lethal test of mice, shows the its effect is raised to 1:64, also approaching document value, LD50>300 LD50/ml, Disc-PAGE shows it has a single cont-eni, which is composed by 17 Amino acid includin asparagic acid.