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目的：制备绿脓杆菌外毒素Ａ（ＰＥＡ）抗原。方法：用胰酶酪蛋白大豆肉汤（Ｔｒｙｐｔｉｃｓｏｙｂｒｏｔｈ，ＴＳＢ），于３２℃水浴１２０次／ｍｉｎ振荡培养绿脓杆菌ＰＡ１０３株，其培养物经７０００ｒ／ｍｉｎ离心，取上清用７０％饱和硫酸铵沉淀，ＳｅｐｈａｄｅｘＧ－１００柱层析，ＤＥＡＥ－５２（ＤＥ－５２）柱层析，羟基磷灰石柱层析制备ＰＥＡ。结果：纯化的ＰＥＡ经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）为一条带，分子量为Ｍｒ（６６～６８）×１０３ｕ。环状沉淀试验，琼脂双向扩散试验，酶联免疫吸附试验和免疫印迹分析表明本室纯化的ＰＥＡ，抗原性和分子量均与文献报道一致。结论：用本方法制备ＰＥＡ，避免了对ＤＥ－５２的反复再生，缩短了实验流程，对保持ＰＥＡ的生物活性有利。
Objective: To prepare Pseudomonas exotoxin A (PEA) antigen. Methods: Pseudomonas aeruginosa strain PA103 was cultured by shaking with Tryptic sobroth (TSB) at 32 ℃ for 120 times / min and the culture was centrifuged at 7000 r / min. The supernatant was extracted with 70% saturated ammonium sulfate Precipitation, SephadexG-100 column chromatography, DEAE-52 (DE-52) column chromatography, hydroxyapatite column chromatography to prepare PEA. Results: Purified PEA was a band with molecular weight of Mr (66-68) × 103u by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Cyclic sedimentation test, agar diffusion test, ELISA and Western blot analysis showed that purified PEA, antigenicity and molecular weight were reported in the literature. Conclusion: The preparation of PEA by this method can avoid repeated regeneration of DE-52 and shorten the experimental procedure, which is beneficial to maintain the bioactivity of PEA.