具有抗血小板聚集功能的新型葡激酶突变体的表达、纯化及性质

目的]为解决溶栓后再栓塞问题,构建N-端含RGD(Arg-Gly-Asp)序列的葡激酶双功能突变体.研究突变体的表达和纯化,并进行性质分析.方法]将突变后的葡激酶突变体序列连入pBV220质粒,转化大肠杆菌BL21进行表达.阳离子交换、凝胶过滤和阴离子交换三步层析法纯化表达产物,采用溶圈法对纯化产物进行生物学活性测定,并测定纯化产物对血小板聚集的抑制效应.结果]PAGE扫描结果显示,葡激酶突变体蛋白在大肠杆菌BL21中的表达量约占菌体蛋白总量的40%～50%;三步层析纯化后,HPLC测定其纯度可达95%.酪蛋白凝胶板溶圈法测得其比活性分别为10.8×104和11.0×104HU/mg,与野生型葡激酶活性相当;且具有明显的抗血小板聚集活性,血小板聚集仪测定其血小板聚集抑制率分别为10.72%和19.71%,明显高于野生型葡激酶血小板聚集抑制率.本实验利用pBV220载体高效表达了葡激酶突变体基因,得到了高纯度、高活性的突变体蛋白,为葡激酶生产产业化和临床应用奠定了良好的基础.

Expression, purification and characterization of a novel staphylokinasewith anti-platelet aggregation activity

Objective] To investigate construction and expression of novel staphylokinase (SAK) mutants with Arg-Gly-Asp (RGD) motif at its N-terminus, and to develop a novel thrombolytic agent with fibrinolytic activity and anti-platelet activity. Methods] The fragments of SAK mutants were inserted into expression plasmid pBV220 and recombinant vectors were transformed into E. coli BL21, The SAK mutants could be expressed by rose of the culture temperature from 30°C to 42°C. After purified by a 3-step chromatography, their fibrinolytic and anti-platelet aggregation activity were analyzed. Results] SDS-PAGE analysis indicated that the SAK mutants occupied 50% of the total proteins in E coli BL21. After purified by the 3-step chromatography, the purity of SAK mutants were more than 95%. The Specific fibrinolytic activities of SAK mutants purified were 10.8×104 and 11.0×104 HU·mg-1.Anti-platelet aggregation activity were 10.72% and 19.71%, which were significantly higher than that of wild-type SAK. Thus the SAK variants were successfully expressed, and purified. The high purity and bioactivities of staphylokinase variants lay a good basis for manufacture and clinical application.