葡激酶聚乙二醇修饰及活性鉴定研究

发布时间：2018-01-16 09:16

本文关键词：葡激酶聚乙二醇修饰及活性鉴定研究　出处：《重庆医科大学学报》2017年07期 　论文类型：期刊论文

【摘要】：目的:通过对葡激酶(staphylokinase,SAK)基因序列进行定点突变,表达突变蛋白,并对其进行聚乙二醇(polyethylene glycol,PEG)修饰。通过分子筛分离得到较高纯度修饰蛋白,在体内外鉴定其溶栓活性。方法:根据SAK晶体结构及抗原位点选择突变位点,构建突变质粒,并在大肠杆菌中表达半胱氨酸(cysteine,Cys)突变蛋白S-cys;分离纯化目的蛋白;对目的蛋白在不同条件下进行聚乙二醇修饰,筛选最优修饰条件;分离修饰蛋白与未修饰蛋白,得到较高纯度的修饰蛋白peg-S-cys,通过纤维蛋白平板溶圈实验和动物栓塞模型的构建初步对其生物活性进行验证。结果:原核表达、纯化后得到纯度较高的S-cys突变体蛋白;优化筛选S-cys蛋白的PEG修饰实验参数,得到最佳修饰条件;应用分子筛分离纯化,成功获得纯度较高的peg-S-cys蛋白(纯度大于90%);初步验证peg-S-cys具有溶栓活性。结论:通过对蛋白质基因序列的定点突变,成功实现了蛋白质的聚乙二醇定点修饰,在体外和体内初步验证其活性,为蛋白质改造提供了一种新的方法与思路。
[Abstract]:Objective: to express the mutant protein by site-directed mutation of staphylokinase staphylokinase (staphylokinase) SAK gene sequence. The modified protein was modified by polyethylene glycol polyethylene (PEG) and purified by molecular sieve. Methods: according to the crystal structure and antigenic site of SAK, the mutant plasmid was constructed, and cysteine was expressed in Escherichia coli. Cys) mutant protein S-cys. Isolation and purification of the target protein; The target protein was modified with polyethylene glycol under different conditions. High purity modified protein peg-S-cys was obtained by isolating modified protein and unmodified protein. The biological activity was preliminarily verified by fibrinolysis assay and animal embolization model. Results: the purified S-cys mutant protein was purified and expressed in prokaryotic expression. The PEG modification parameters of S-cys protein were optimized and the optimum modification conditions were obtained. Peg-S-cys protein with high purity was successfully obtained by molecular sieve separation and purification. Conclusion: through the site-directed mutation of protein gene sequence, the protein was successfully modified with pegyleneglycol. Its activity was preliminarily verified in vitro and in vivo, which provided a new method and train of thought for protein modification.
【作者单位】：重庆医科大学附属第一医院心血管内科;重庆医科大学感染性疾病分子生物教育部重点实验室;
【分类号】：R3411
【正文快照】： 蛋白质改造即通过对蛋白质化学、蛋白质晶体学和蛋白质动力学的研究,获得有关蛋白质理化特性和分子特性的信息,在此基础上对编码蛋白质的基因进行有目的的设计和改造[1]。目前蛋白质改造的手段主要有定点突变、融合蛋白、功能域表达、蛋白修饰等。研究表明,通过对蛋白质进行聚

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