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目的:对水蛭活性肽进行纯化,并对水蛭活性肽进行树脂纯化条件考察。方法:采用DA201-C大孔吸附树脂进行极性分离,后经D201阴离子交换树脂进行电荷分离,最终得到纯化的水蛭活性肽组分。结果:DA201-C大孔树脂,上样肽浓度20 mg·m L~(-1),流速1.0 BV﹒h~(-1),依次用25%乙醇、50%乙醇和75%乙醇溶液洗脱,各洗脱部位得率及活力较高,但总体活性成分被分散,以上各洗脱部位经D201离子交换树脂,p H 4.0,上样肽浓度30 mg﹒m L~(-1),流速3.0 BV·h~(-1),分别用2.5%氯化钠的25%乙醇、2.5%氯化钠的50%乙醇、2.5%氯化钠的75%乙醇溶液洗脱,2.5%氯化钠的25%乙醇和2.5%氯化钠的50%乙醇洗脱部位有明显的抗凝活性,经过分析性RP-HPLC验证后,基线平稳,各组分分离度良好。结论:经DA201-C大孔树脂和D201离子交换树脂纯化后的水蛭活性肽,2.5%氯化钠的25%乙醇和2.5%氯化钠的50%乙醇洗脱部位活力较高,分离度好,可为进一步的制备合成奠定基础。
OBJECTIVE: To purify leptin active peptide and investigate the conditions of lepirin active peptide purification. Methods: DA201-C macroporous adsorption resin was used for polar separation. After D201 anion exchange resin was used for charge separation, the purified leech peptide was obtained. Results: The macroporous resin DA201-C was loaded with a peptide concentration of 20 mg · mL -1 at a flow rate of 1.0 BV. h ~ (-1), followed by elution with 25% ethanol, 50% ethanol and 75% ethanol, the yield and activity of each eluted site were higher, but the total active components were dispersed. Exchange resin, p H 4.0, loading peptide concentration 30 mg. m L -1, the flow rate was 3.0 BV · h -1, respectively, with 2.5% sodium chloride in 25% ethanol, 2.5% sodium chloride in 50% ethanol, 2.5% sodium chloride in 75% Solution elution, 2.5% sodium chloride 25% ethanol and 2.5% sodium chloride 50% ethanol elution part of significant anticoagulant activity, after the analysis of RP-HPLC verification, the baseline was stable, the resolution of each component good. CONCLUSION: The bioactive peptides with 2.5% sodium chloride, 25% ethanol and 2.5% sodium chloride in 50% ethanol have the highest activity and good resolution after purified by DA201-C macroporous resin and D201 ion exchange resin , Which can lay the foundation for further preparation and synthesis.