葡激酶聚乙二醇修饰及活性鉴定研究  

Study on staphylokinase pegylation and preliminary function

作　　者：白垒 徐彦颖[1] 汪德强[2] 周建中[1] 

机构地区：[1]重庆医科大学附属第一医院心血管内科 [2]重庆医科大学感染性疾病分子生物教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2017年第7期893-897,共5页Journal of Chongqing Medical University

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。Objective：To obtain a site-specific pegylated protein by site-specific mutagenesis to staphylokinase（SAK） protein gene sequence and to progres polyethylene glycol（PEG） on mutant protein on variant circumstances;to obtain modified protein with high purity by selecting the optimum modification condition and using molecular sieve and to identify its function in vivo and in vitro. Methods：Primer was designed according to＇ the crystal structure and antigen sites of SAK protein;the selected amino acid on SAK protein was mutant into cysteine（Cys） by PCR. Mutant plasmid was obtained and transformed into BL21 （DE3） cell. Mutant protein S-cys was expressed in escherichia coli cells through prokaryotic expression technology. Purified mutant protein was obtained. Pegy- lation was progressed under variant circumstances, and optimum modification condition was screened. Higher purity of pegylated pro- tein peg-S-cys was obtained through molecular sieve. Biological activity was preliminary validated by fibrin-agarose plate process and animal embolism model. Results： Cys on target site was obtained successfully. High purity of S-cys protein was obtained. S-cys protein was modified, and optimal modify condition was screened. High purity of peg-S-cys protein was obtained. Thrombolytic a- bility of peg-S-cys was confirmed preliminary. Conclusion：Site-specific pegylated protein can be successfully obtained, and the re- constructed protein is biologicallv active in vitro and in vivo.

关 键 词：蛋白质改造 聚乙二醇修饰 定点突变 活性验证 

分 类 号：Q816[生物学—生物工程]