摘要
目的优化聚乙二醇(polyethylene glycol,PEG)化重组人铜锌超氧化物歧化酶(hCu,Zn-SOD)改构体(rmhCu,Zn-SOD)的制备工艺,以期延长其在体内的半衰期。方法以相对分子质量5 000的甲氧基聚乙二醇乙酸-羟基琥珀酰亚胺酯(mPEG-SPA)作为修饰剂,对rmhCu,Zn-SOD蛋白进行化学修饰,并对mPEG-SPA与rmhCu,Zn-SOD的摩尔比、反应缓冲液、反应温度、反应方式及反应时间进行优化;利用硫酸铵沉淀、凝胶过滤色谱法对PEG化rmhCu,Zn-SOD进行分离纯化,并检测其在小鼠体内的半衰期。结果 PEG化rmhCu,Zn-SOD的最佳制备工艺为mPEG-SPA与rmhCu,Zn-SOD的摩尔比1.5∶1,缓冲液为pH 8.5的磷酸盐缓冲液,反应液混匀后4℃静置反应1 h;纯化的修饰产物的纯度约为90%,残余酶活力为(84.3±2.3)%,蛋白修饰率为(65.4±1.9)%;与未修饰的rmhCu,Zn-SOD相比,PEG化rmhCu,Zn-SOD的半衰期延长了约7倍。结论优化了PEG化rmhCu,Zn-SOD的制备工艺;PEG化修饰有效延长了该改构体蛋白在体内的半衰期。
Objective To optimize the preparation procedure of PEGylated recombined mutant human Cu,Zn-SOD（rmhCu, Zn-SOD）so as to increase its half-life in vivo. Methods The rmhCu,Zn-SOD was modified chemically with monomethoxypoly（ethylene glycol）succinimidyl propionate（mPEG-SPA）with a relative molecular mass of 5 000,and the molar ratio of mPEG-SPA to rmhCu,Zn-SOD as well as reaction buffer,reaction temperature,reaction mode and reaction time were optimized. The PEGylated rmhCu,Zn-SOD was isolated and purified by ammonium sulfate precipitation and gel filtration chromatography,and determined for half-life in vivo in mice. Results The optimal molar ratio of mPEG-SPA to rmhCu, Zn-SOD was 1. 5 ∶ 1,while the optimal buffer was phosphate buffer at a pH value of 8. 5. The reaction solutions were mixed up,allow to stand at 4 ℃ for 1 h. The purity,residual enzyme activity and protein modification rate of PEGylated rmhCu,Zn-SOD were about 90%,（84. 3 ± 2. 3）% and（65. 4 ± 1. 9）% respectively,while the half-life increased by 7 folds compared with that unmodified. Conclusion The preparation procedure of PEGylated rmhCu,Zn-SOD was optimized,and the half-life in vivo of PEGylated rmhCu,Zn-SOD increased significantly.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第2期245-250,共6页
Chinese Journal of Biologicals
基金
陕西省科学院重点项目和产业化专项(2011K-04)
中国科学院"西部之光"人才培养项目(2009DF03)
