【作者】 陈佳乐；

【导师】 应国清；

【作者基本信息】 浙江工业大学 ， 生物化工， 2008， 硕士

【摘要】 蛋白质分子修饰是蛋白类药物二次开发中的一项热点技术,可解决诸多蛋白类药物的应用问题,如稳定性差,半衰期短等,但修饰过程一般复杂,操作步骤多、周期长。本文以超氧化物歧化酶（Superoxide dismutase,SOD）为模板蛋白,探讨将分离过程和修饰过程耦合起来,从而获得活性高,稳定性更好的修饰SOD,并以此探求蛋白分离过程和修饰过程耦合的可行性。本文首先以DEAE 52为层析介质,研究了SOD阴离子交换吸附分离-PEG修饰耦合过程。研究结果表明,SOD最佳吸附条件:在pH7.8,温度25℃,溶液浓度1mg/ml、的吸附条件下,SOD吸附量达到5.3366x10⁴U/g;固相SOD的最佳修饰条件:在25℃,pH9.2,50mmol/g硼砂-硼酸缓冲液中,mPEG与SOD的摩尔比为50:1;修饰后SOD的最佳洗脱条件:0-0.4 M NaCl梯度洗脱,流速为0.96mL/cm²·min。最终获得PEG-SOD的比活力:9638 U/mg,纯化倍数1.87倍,氨基修饰率:35.3%。然后,以自制Cu²⁺-葡聚糖凝胶G-75为层析介质,研究SOD金属螯合吸附分离-PEG修饰耦合过程。研究结果表明,SOD最佳吸附条件:在pH8.0,温度25℃,溶液浓度1.5mg/ml,Nacl浓度0.5M的吸附条件下,SOD吸附量达到9.69x10⁴ U/g;固相SOD的最佳修饰条件:在25℃,pH9.2,100 mmol/L硼砂-硼酸缓冲液中,mPEG与SOD摩尔比为50:1;修饰后SOD的最佳洗脱条件:pH7.8,,25mmol/L磷酸缓冲液（0.5mol/L Nacl）洗脱杂蛋白后,再用pH6.0,25mmol/L磷酸盐缓冲液（0.5 mol/L Nacl,0.5 mol/L的NH₄C1）洗脱SOD。最终获得PEG-SOD的比活:9067.8 U/mg,纯化倍数:1.755倍;氨基修饰率:40.9%,相比等量游离SOD的层析产品,活性保留:88.43%。最后,考察了修饰后SOD的部分性质,结果发现经修饰后的SOD稳定性获得了提高;同时通过对SOD新的修饰过程探讨,进一步论证了蛋白质吸附分离-化学修饰过程耦合的可行性。更多还原

【Abstract】 Molcular modification technology is one of the hotspots in the second development of protein drugs, which can solve lots of problems in applying the drugs, such as unstability and short half-life. However, there are also some deficiencies of this technology, the process is complicated with many steps and long periods. In this paper, SOD was used as a template, and the procedures of purification and modification were coupled to obtain more stable modified SOD with higher activity. And moreover, the feasibility of purification-modification coupling process in separating proteins was discussed.First of all, SOD’s coupling procedure of IEC purification - PEG modification was studied, using DEAE 52 as solid phase medium. The adsorption and modification condition was optimized by orthogonal design. Absorption temperature was 25℃, pH was 7.8, solution concentration was 1.0 mg/ml. The adsorption capacity reached 5.3366×10⁴U/mg. SOD modified by mPEG（50 fold）, at temperature 25℃, in borate buffer（ pH 9.2, 50 mmol/L）. PEG-SOD washed by NaCl （0-0.4 M） grads. As a result, the specific activity of PEG-SOD is 9 638 U/mg, with purification rate of 1.87. It was modified at 35.3% of its free amino groups.Secongdly, SOD’s coupling procedure of MCAC purification - PEG modification was studied, using Cu²⁺-sephadex G75 as solid phase medium. The adsorption and modification condition was optimized by orthogonal design. Absorption temperature was 25℃, pH was 8.0, solution concentration was 1.5 mg/ml and contained 0.5 M NaCl. The adsorption capacity reached 9.69×10⁴ U/g. SOD modified by mPEG（50 fold）, at temperature 25℃, in borate buffer（pH 9.2, 100 mmol/L）. Then PEG-SOD was eluted by Tris-HCl buffer （pH6.0, 0.5 M NH₄Cl）. As a result, the specific activity of PEG-SOD is 9 067 U/mg, with purification rate of 1.755. It was modified at 40.9% of its free amino groups, retained 88.43% of enzymatic activity of native SOD.Finally, partial properties of modified SOD were studied. The results showed that the stability of PEG-SOD was enhanced significantly and we found it feasible to couple purification process with modification process in separating process.更多还原