精氨酸脱亚胺酶的PEG修饰及其药理学研究
发布时间：2018-11-13 10:05
【摘要】：精氨酸脱亚胺酶(Arginine deiminase，ADI，EC3.5.3.6)能将L-精氨酸不可逆地水解为L-瓜氨酸和氨，可用于治疗精氨酸营养缺陷型癌症。PEG修饰蛋白质药物可以提高蛋白药物的半衰期和药理学特性，并可获得更加良好的疗效和较低的副作用。为解决ADI在体内生理条件下的半衰期短和免疫原性强等问题，本研究采用PEG对ADI进行修饰并研究其药理学特性。 在前期研究中，通过蛋白质工程改造获得了一株来源于变形假单胞菌(Pseudomonasplecoglossicida)，在生理条件下具有高比酶活和改良的酶学性质的ADI优良突变株M13-3。首先，采用单因素优化与响应面法优化M13-3菌株发酵培养基，提高产酶效率。优化后培养基经摇瓶发酵，ADI酶活达到5.7U mL-1发酵液，比LB培养基提高了2.01倍。在3L发酵罐中发酵8h后，ADI酶活最大值为17.38U mL-1发酵液，较LB培养基提高了3.51倍。 对M13-3菌株发酵后的粗酶液进行阴离子交换层析和凝胶过滤层析纯化获得ADI纯酶，选用七种不同PEG修饰剂对ADI进行修饰，获得了三种产率较高的修饰产物：ADI-SS-PEG20kDa，ADI-SC-PEG20kDa，ADI-SPA-PEG20kDa。经过体外稳定性分析，三种PEG修饰酶较好地保留了ADI在生理条件下的酶活，且pH稳定性和热稳定性都略有提高。 选择ADI-SPA-PEG20kDa(ADI-SPA)进行药理学实验，，由药效/药代动力学结果分析可得，ADI-SPA修饰酶在小鼠体内表现出增强的药物效应与延长的药效时间，5U注射剂量均能有效降解血液中精氨酸并维持5天以上，大大高于未修饰的ADI。药代动力学参数整体上也优于未修饰酶，其中经由静脉注射比肌肉注射和皮下注射具有更好的药代动力学表现：静脉注射5UADI-SPA，半衰期为53.2h，较ADI提高了11倍；而通过肌肉注射则为34.5h，提高了6倍多。 在H22肿瘤模型小鼠体内对ADI-SPA进行药物评估，结果显示通过肌肉注射高剂量组ADI-SPA，即每5天注射5U ADI-SPA，15天内共注射3次共计15U，对H22肝癌的抑制率可以达到95.02%，高于ADI-SPA中、低剂量组和ADI组，说明总剂量相同的ADI-SPA比未修饰ADI具有更强更持久的疗效。同时高剂量ADI-SPA组抑制率接近5-FU化疗药物治疗的阳性组(98.34%)。本研究结果表明，PEG修饰的ADI酶(如：ADI-SPA)可以作为有效的抗肝癌药物进行进一步研究。
[Abstract]:Arginine deiminase (Arginine deiminase,ADI,EC3.5.3.6) can hydrolyze L-arginine irreversibly into L-citrulline and ammonia. PEG modified protein drugs can improve the half-life and pharmacological properties of protein drugs and obtain better curative effect and lower side effects. In order to solve the problems of short half-life and strong immunogenicity of ADI under physiological conditions in vivo, PEG was used to modify ADI and its pharmacological properties were studied. In the previous study, we obtained an excellent mutant M13-3 of ADI derived from Pseudomonas mutans (Pseudomonasplecoglossicida), with high specific enzyme activity and improved enzymatic properties under physiological conditions. First, single factor optimization and response surface method were used to optimize the fermentation medium of strain M13-3 to improve the efficiency of enzyme production. After fermentation in shaking flask, the enzyme activity of ADI reached 5.7 U mL-1 fermentation broth, which was 2.01 times higher than that of LB medium. After 8 h fermentation in a 3L fermenter, the maximum activity of ADI was 17.38 U mL-1 fermentation broth, which was 3.51 times higher than that of LB medium. ADI pure enzyme was purified by anion exchange chromatography and gel filtration chromatography from the fermented crude enzyme solution of strain M13-3. Seven different PEG modifiers were used to modify ADI. Three modification products with higher yield were obtained: ADI-SS-PEG20kDa, ADI-SC-PEG20kDa,ADI-SPA-PEG20kDa. After in vitro stability analysis, the three PEG modifiers retained the enzyme activity of ADI under physiological conditions, and the stability and thermal stability of pH were improved slightly. ADI-SPA-PEG20kDa (ADI-SPA) was selected for pharmacological experiment. The results of pharmacodynamics / pharmacokinetics showed that ADI-SPA modified enzyme showed enhanced drug effect and prolonged pharmacodynamic time in mice. The injection dose of 5U could effectively degrade arginine in blood for more than 5 days, which was much higher than that of unmodified ADI.. The pharmacokinetic parameters were also better than that of unmodified enzyme on the whole. The pharmacokinetics of 5UADI-SPA by intravenous injection was better than that by intramuscular injection and subcutaneous injection. The half-life of 5UADI-SPA was 53.2 h, which was 11 times higher than that of ADI. But the intramuscular injection was 34.5 h, which increased by more than 6 times. ADI-SPA was evaluated in H22 tumor model mice. The results showed that ADI-SPA, was injected three times every 5 days with 5 U ADI-SPA,15 in high dose group. The inhibition rate of H22 liver cancer was 95.02, which was higher than that of ADI-SPA, low dose group and ADI group, indicating that ADI-SPA with the same total dose had stronger and more lasting curative effect than unmodified ADI. At the same time, the inhibitory rate of high dose ADI-SPA group was similar to that of 5-FU positive group (98.34%). The results showed that PEG modified ADI enzyme (such as ADI-SPA) could be used as an effective antitumor drug for further study.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
本文编号：2328774
[Abstract]:Arginine deiminase (Arginine deiminase,ADI,EC3.5.3.6) can hydrolyze L-arginine irreversibly into L-citrulline and ammonia. PEG modified protein drugs can improve the half-life and pharmacological properties of protein drugs and obtain better curative effect and lower side effects. In order to solve the problems of short half-life and strong immunogenicity of ADI under physiological conditions in vivo, PEG was used to modify ADI and its pharmacological properties were studied. In the previous study, we obtained an excellent mutant M13-3 of ADI derived from Pseudomonas mutans (Pseudomonasplecoglossicida), with high specific enzyme activity and improved enzymatic properties under physiological conditions. First, single factor optimization and response surface method were used to optimize the fermentation medium of strain M13-3 to improve the efficiency of enzyme production. After fermentation in shaking flask, the enzyme activity of ADI reached 5.7 U mL-1 fermentation broth, which was 2.01 times higher than that of LB medium. After 8 h fermentation in a 3L fermenter, the maximum activity of ADI was 17.38 U mL-1 fermentation broth, which was 3.51 times higher than that of LB medium. ADI pure enzyme was purified by anion exchange chromatography and gel filtration chromatography from the fermented crude enzyme solution of strain M13-3. Seven different PEG modifiers were used to modify ADI. Three modification products with higher yield were obtained: ADI-SS-PEG20kDa, ADI-SC-PEG20kDa,ADI-SPA-PEG20kDa. After in vitro stability analysis, the three PEG modifiers retained the enzyme activity of ADI under physiological conditions, and the stability and thermal stability of pH were improved slightly. ADI-SPA-PEG20kDa (ADI-SPA) was selected for pharmacological experiment. The results of pharmacodynamics / pharmacokinetics showed that ADI-SPA modified enzyme showed enhanced drug effect and prolonged pharmacodynamic time in mice. The injection dose of 5U could effectively degrade arginine in blood for more than 5 days, which was much higher than that of unmodified ADI.. The pharmacokinetic parameters were also better than that of unmodified enzyme on the whole. The pharmacokinetics of 5UADI-SPA by intravenous injection was better than that by intramuscular injection and subcutaneous injection. The half-life of 5UADI-SPA was 53.2 h, which was 11 times higher than that of ADI. But the intramuscular injection was 34.5 h, which increased by more than 6 times. ADI-SPA was evaluated in H22 tumor model mice. The results showed that ADI-SPA, was injected three times every 5 days with 5 U ADI-SPA,15 in high dose group. The inhibition rate of H22 liver cancer was 95.02, which was higher than that of ADI-SPA, low dose group and ADI group, indicating that ADI-SPA with the same total dose had stronger and more lasting curative effect than unmodified ADI. At the same time, the inhibitory rate of high dose ADI-SPA group was similar to that of 5-FU positive group (98.34%). The results showed that PEG modified ADI enzyme (such as ADI-SPA) could be used as an effective antitumor drug for further study.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
【参考文献】
相关期刊论文 前9条
1 施险峰;詹家荣;廖本仁;;聚乙二醇修饰技术研究进展[J];上海化工;2007年12期
2 郝学财;余晓斌;刘志钰;王蓓;;响应面方法在优化微生物培养基中的应用[J];食品研究与开发;2006年01期
3 段学辉;颜淑芳;彭云召;贾奎艳;;响应面法优化灰绿青霉Penicillium glaucum NS16产酶条件[J];食品与生物技术学报;2010年03期
4 黄雪莲;于新;;响应面优化绿色木霉菌培养基[J];食品与生物技术学报;2011年05期
5 马金波,郄建坤,刘克良;多肽蛋白质类药物聚乙二醇化修饰研究进展[J];中国药物化学杂志;2005年02期
6 王俊,裴元英;用聚乙二醇修饰大分子药物的研究进展[J];中国医药工业杂志;2004年11期
7 肖永红;抗菌药物的药代动力学/药效学概念及其临床意义[J];中华医学杂志;2004年22期
8 赵春海,阚振荣;无机盐及微量元素对乙酰乳酸脱羧酶发酵活力的影响[J];中国酿造;2005年06期
9 李建华;李啸;张娅;徐能;姚娟;李知洪;俞学锋;;金属离子对卤醇脱卤酶发酵生产的影响[J];中国酿造;2012年03期
本文编号：2328774
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2328774.html
最近更新
教材专著
