新型聚乙二醇化葡激酶的制备及功能鉴定研究

发布时间：2018-05-25 15:00

本文选题：葡激酶 + 氨基酸特征结构　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的通过对葡激酶(SAK)基因序列进行定点突变、表达、纯化与进行聚乙二醇修饰以获得较高纯度的聚乙二醇化葡激酶(peg-SAK-cys),并对其溶栓活性与免疫原性初步进行验证。方法设计引物将根据SAK晶体结构与预测抗原位点所挑选的82号氨基酸突变为半胱氨酸并将突变质粒通过化学转化进入BL21(DE3)感受态。利用经典原核表达技术表达突变葡激酶蛋白(SAK-cys)。利用镍离子交换柱、分子筛等方法分离纯化SAK-cys蛋白并对其进行聚乙二醇修饰。用纤维蛋白平板溶圈法和血栓弹力图初步对本次实验制备的peg-SAK-cys与课题组以相同方法制备的另外4种peg-SAK-cys的生物活性进行验证。以酶联免疫吸附(ELISA)法评价这些peg-SAK-cys的免疫原性。分析体外实验数据,并对功能评价较高的peg-SAK-cys通过动物栓塞实验模型进一步功能验证。结果成功获得了82号氨基酸突变的葡激酶质粒,表达、纯化了突变蛋白,并进行聚乙二醇修饰获得了第82号葡激酶突变的葡激酶蛋白(peg-SAK-C82A),分离纯化后纯度在总蛋白质量的90%以上。统计分析与计算溶圈实验结果,第97号与104号氨基酸突变的聚乙二醇化葡激酶(peg-SAK-C97A、peg-SAK-C104G)保留了较高的活性;血栓弹力图实验数据也支持了这一结果;免疫原性测定结果提示peg-SAK-C104G的免疫原性显著低于野生型SAK(P=0.0002);评价peg-SAK-C104G对SD大鼠颈动脉栓塞模型的溶栓效果,显示蛋白于体内实验也保留了相当的溶栓能力。结论通过位点特异性特变与聚乙二醇修饰技术的联合运用可以成功改造出有较低免疫原性的活性葡激酶。
[Abstract]:Objective to obtain high purity peg-SAK-cys1 by site-directed mutation, expression, purification and modification of staphylokinase SAK gene, and to verify its thrombolytic activity and immunogenicity. Methods A primer was designed to mutate the 82 amino acid selected according to the crystal structure and predicted antigenic site of SAK into cysteine and the mutant plasmid was chemically transformed into BL21DDE3). Classical prokaryotic expression technique was used to express the mutant staphylokinase protein SAK-cys1. SAK-cys protein was purified by nickel ion exchange column and molecular sieve and modified with polyethylene glycol. The bioactivity of peg-SAK-cys prepared in this experiment was preliminarily verified by fibrin plate method and thromboelastic diagram with the other four kinds of peg-SAK-cys prepared by the same method. Enzyme linked immunosorbent assay (Elisa) was used to evaluate the immunogenicity of these peg-SAK-cys. The experimental data in vitro were analyzed and the peg-SAK-cys with high function evaluation was further verified by animal embolization model. Results the staphylokinase plasmid with 82 amino acid mutation was successfully obtained, the mutant protein was expressed and purified, and the mutant protein peg-SAK-C82 was obtained by polyethylene glycol modification. The purity of the mutant protein was more than 90% of the total protein content. The results of statistical analysis and calculation showed that peg-SAK-C97Apeg-SAK-C104G) with amino acid mutations of 97 and 104 amino acids retained high activity, and the experimental data of thromboelastic diagram also supported the results. The results of immunogenicity test showed that the immunogenicity of peg-SAK-C104G was significantly lower than that of wild-type SAKP 0.0002, and the thrombolytic effect of peg-SAK-C104G on carotid artery embolization model of SD rats was evaluated, which showed that the protein also retained considerable thrombolytic ability in vivo. Conclusion the staphylokinase with low immunogenicity can be successfully modified by the combination of site-specific mutagenesis and polyethylene glycol modification.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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