重组水蛭素的聚乙二醇修饰

重组水蛭素的聚乙二醇修饰

李雪芹，候蓓蓓，赵军，田明玉，修志龙*

XX理工大学环境与生命学院XX 116024

摘要：目的水蛭素因血浆半衰期短而严重限制了其临床应用，聚乙二醇修饰能有效地延长其半衰期。本文通过比较不同修饰位点、修饰方法所得单修饰产物的比率、纯度及其活性保留率，从而得到修饰专一性强且活性保留率较高的修饰策略。方法采用液相和固相修饰方法,用琥珀酰亚胺活化的PEG 20kDa分别在pH6.0、8.0的条件下对水蛭素的His和Lys进行定点修饰；用SDS-PAGE分析产物的修饰度，并用离子交换柱对修饰后的产物进行分离，然后用凝血酶滴定法测定水蛭素单修饰产物的体外抗凝活性。另外用分子动力学模拟方法预测了pH8.0条件下PEG修饰的位点。结果在pH6.0、8.0的条件下，水蛭素单修饰率都高达90%以上，但二者的单修饰活性保留率相差较大。在pH6.0时，液、固相单修饰活性保留率为34%、34.8%；而在pH8.0时分别为55%、96%。结论在pH8.0 条件下，采用“离子交换柱辅助”固相修饰的方法对Lys进行定点修饰能得到较高产率和较高活性保留率的单修饰产物。关键词：重组水蛭素；SC-mPEG；抗凝活力；分子动力学模拟

PEGylation of Rebinant Hirudin

Li Xueqin，Hou Beibei，Zhao Jun，Tian Mingyu，Xiu Zhilong* Department of Bioscience and Biotechnology,School of Environmental and Biological Science and Technology,DalianUniversity of Technology,Dalian 116024,China

Abstract:OBJECT Hirudin is the most potent inhibitor of thrombin found in nature. Although hirudin has the strongest anti-thrombin activity in vivo, its short half-life in serum significantly limits its clinical anticoagulant application. Currently, PEGylation is monly used as an effective method to prolong its half-life in serum.Our object of experiment is to choose the best PEGylation method according to the monoPEGylated rebinant h irudin’s purity and the anticoagulant activity in vitro.METHODS Solution method and solid method assisted by “packed-bed” and anion exchange columnwere used to favor the formation of mono-PEGylated hirudin. The mild acidic and mild alkaline

PEGylation strategy was used to target His residue and Lysresidue of hirudinrespectively using SC-mPEG20kDa.The pegylated productswere isolated by anion exchange chromatogram. SDS-PAGE was used to analyze the purity of PEGylated hirudin. Thrombin method was used to analyze the anticoagulant activity of PEGylatedhirudin. Molecular dynamics modeling was used to judge the probability of PEGylation site. RESULTS The mono-PEGylated product with a purity of more than 90% was obtained at pH6.0 and pH8.0. But a large difference in anticoagulant activity exists: the anticoagulant activity of solution and solid methods were 34% and 34.8% respectively at pH6.0；55%、96% at pH8.0.CONCLUSION Solid method assisted by anion exchange column at pH8.0 was a better strategy to get mono-PEGylated r-Hirudin.

Key words：rebinant hirudin；SC-mPEG；anticoagulant activity；molecular dynamics modeling

前言

水蛭素是含65个氨基酸残基与3个二硫键的多肽，分子量约7000，是迄今为止发现的特异性最好的凝血酶抑制剂[1],而凝血酶诱发的血液凝固是诱导血管血栓形成的重要原因，因此水蛭素对各种血栓病均有疗效。

然而水蛭素有一些显著的药用缺点，如血浆半衰期较短，一般只有60-100分钟，患者需不断注射才能维持抗凝效果，导致治疗成本较高，且重复注射也会引起一些不良反应[2-4]。上世纪70年代以来，人们发现一些蛋白经过PEG修饰后，很多方面的药用特性大大改善。之后，人们开展了对于水蛭素的PEG修饰研究。George C.Avgerinos[5]等人采用基因工程方法对水蛭素氨基酸进行改造，用甲基营养酵母Hansenyla polymorpha特异性表达了只含两个Lys的水蛭素，采用对硝基碳酸酯活化的PEG 5kDa进行修饰，并设计了工业等级纯化步骤。这种对水蛭素修改的方式可以大大提高修饰产物的专一性，但是研究周期性较长且须具备一定的科研条件。

国内也有关于水蛭素PEG修饰的研究。于爱平[6]等人采用SPA-PEG 5kDa修饰水蛭素II，采用Source Q15离子交换柱凝胶柱分离修饰产物，发现三修饰产物活力大大降低，为原蛋白的33.5%。秦海娜[7]等人采用羰基二咪唑法活化的PEG 5kDa修饰水蛭素，采用凝胶色谱方法X修饰产物进行分离，虽水蛭素可与修饰产物分离，但是修饰产物之间未能分离开。