新型聚乙二醇化葡激酶的表达、纯化及功能鉴定研究  

Expression, purification and characterization of pegylation staphylokinase

作　　者：徐彦颖[1] 白垒 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内科,重庆400016 [2]重庆医科大学检验医学院,重庆400016

出　　处：《中国新药与临床杂志》2017年第4期203-209,共7页Chinese Journal of New Drugs and Clinical Remedies

基　　金：重庆市科委资助项目(cstc2012gg-gjhz0025)

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。方法根据SAK蛋白晶体结构及抗原位点选择突变位点,设计引物将所选的氨基酸突变为半胱氨酸。将突变质粒通过化学转化进入BL21(DE3)感受态,并利用经典原核表达技术,在大肠杆菌中表达突变葡激酶(SAK-cys)。利用镍离子交换柱、分子筛等方法分离纯化目的蛋白。用纤维蛋白平板溶圈法和血栓弹力图初步对其生物活性进行验证。以酶联免疫吸附(ELISA)法评价peg-SAK-cys的免疫原性。结果成功获得了SAK-cys质粒,表达、纯化了突变蛋白,并进行聚乙二醇修饰获得了peg-SAK-cys,分离纯化后纯度在总蛋白质量的90%以上。计算其溶圈实验结果,活性为8.2×10~4IU·mg^(-1);血栓弹力图实验结果提示其具有较高的溶栓活性;免疫原性测定结果提示peg-SAK-cys免疫原性低于野生型SAK(P=0.000 2)。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SAK。AIM The staphylokinase （SAK） gene sequence was mutated by the site-specific mutation technology, and the PEGylated SAK protein was progressed by expression, PEGylation, and purification consequently. The biological activity and immunogenicity of the PEGylated SAK had been measured. METHODS Primers was designed according to the crystal structure and antigen sites of SAK. The selected amino acid was mutated into cysteine by PCR. The mutant gene sequence was transformed into BL21 （DE3） cell and expressed with classic prokaryotic expression technology. The mutant SAK which had been expressed was purified by NiNTA sepharose. The higher purity PEGylated SAK was obtained by PEGylation and secondary separation. Soluble fibrin plate method and thromboelastogram （TEG） was used to characterize the fibrinolytic activity of PEGylated SAK. Besides, the immunogenicity of SAK was measured by Enzyme-linked immunosorbent assay （ELISA）. RESULTS The mutant plasmid was constructed successfully. The mutant SAK protein was expressed and purified well, and more than 90% purity of the PEGylated SAK was obtained by PEGylation and second purification. The fibrin plate assay revealed that the fibrinolytic activity of PEGylated SAK was 8.2 × 10 4 IU ·mg-1, similar results were observed in the thromboelastogram experiment. The enzyme- linked immune- sorbent assay revealed that the immunogenicity of PEGylated SAK was lower than wild-type SAK （P = 0.000 2）. CONCLUSION The SAK, with low fibrinolytic activity and low imunogenicity, can be successfully produced by site- specific mutation and PEGylation technology.

关 键 词：葡激酶 氨基酸特征结构 点突变 聚乙烯二醇类 免疫遗传学 

分 类 号：R973.2[医药卫生—药品]