新型聚乙二醇化葡激酶的表达、纯化及功能鉴定研究
发布时间：2018-11-02 12:06
【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。
[Abstract]:Objective to obtain a high purity (SAK) gene by site-directed mutation, expression, purification and modification with polyethylene glycol (PEG). Its thrombolytic activity and immunogenicity were preliminarily verified. Methods according to the crystal structure and antigenic site of SAK protein mutation sites were selected and primers were designed to mutate the selected amino acids to cysteine. The mutant plasmid was chemically transformed into BL21 (DE3) competent state and expressed in E. coli by classical prokaryotic expression technique. The target protein was purified by nickel ion exchange column and molecular sieve. The bioactivity was preliminarily verified by fibrin plate method and thromboelastic diagram. The immunogenicity of peg-SAK-cys was evaluated by enzyme linked immunosorbent assay (ELISA). Results the SAK-cys plasmid was successfully obtained, the mutant protein was expressed and purified, and the purity of peg-SAK-cys, was more than 90% of the total protein. The results of circle dissolution test showed that the activity was 8.2 脳 10~4IU mg~ (-1), the thromboelastogram showed that the thrombolytic activity was higher, and the immunogenicity of peg-SAK-cys was lower than that of wild type SAK (P0. 0002). Conclusion SAK. with low immunogenicity can be successfully modified by the combination of site-specific mutagenesis and polyethylene glycol modification.
【作者单位】： 重庆医科大学附属第一医院心内科;重庆医科大学检验医学院;
【基金】：重庆市科委资助项目(cstc2012gg-gjhz0025)
【分类号】：R915
，
本文编号：2305962
[Abstract]:Objective to obtain a high purity (SAK) gene by site-directed mutation, expression, purification and modification with polyethylene glycol (PEG). Its thrombolytic activity and immunogenicity were preliminarily verified. Methods according to the crystal structure and antigenic site of SAK protein mutation sites were selected and primers were designed to mutate the selected amino acids to cysteine. The mutant plasmid was chemically transformed into BL21 (DE3) competent state and expressed in E. coli by classical prokaryotic expression technique. The target protein was purified by nickel ion exchange column and molecular sieve. The bioactivity was preliminarily verified by fibrin plate method and thromboelastic diagram. The immunogenicity of peg-SAK-cys was evaluated by enzyme linked immunosorbent assay (ELISA). Results the SAK-cys plasmid was successfully obtained, the mutant protein was expressed and purified, and the purity of peg-SAK-cys, was more than 90% of the total protein. The results of circle dissolution test showed that the activity was 8.2 脳 10~4IU mg~ (-1), the thromboelastogram showed that the thrombolytic activity was higher, and the immunogenicity of peg-SAK-cys was lower than that of wild type SAK (P0. 0002). Conclusion SAK. with low immunogenicity can be successfully modified by the combination of site-specific mutagenesis and polyethylene glycol modification.
【作者单位】： 重庆医科大学附属第一医院心内科;重庆医科大学检验医学院;
【基金】：重庆市科委资助项目(cstc2012gg-gjhz0025)
【分类号】：R915
，
本文编号：2305962
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2305962.html
最近更新
教材专著
