文章编号] 1674-4721（2019）6（c）-0008-06
[Abstract] Objective To prepare the most active recombinant human CYP2C9 yeast microsomes by optimizing the conditions of glass bead crushing method， thus provide and establish more sensitive and rapid in vitro metabolic screening model with the minimum metabolic content degradation. Methods Recombinant CYP2C9 human microsomes content was prepared by glass bead crushing method. By changing the number of vortex oscillations and the amount of glass beads， the conditions of preparing microspheres were optimized. Lowry method was used to determine protein and metabolite concentration in microsomes. The reaction with microsomes was carried out using Diclofenac， which was a typical substrate for 2C9 microsomes. The protein activity of microsomes was determined by the yield of diclofenac metabolite （4-hydroxy diclofenac） corresponding to unit protein （mg/ml） per unit time （h）. Results With the increase of the number of vortex oscillations and the amount of glass beads， the content of the prepared microsomal protein increased. When the number of vortices oscillated 30 times （30 s per vortex oscillation， 60 s of cooling） and the amount of glass beads was 0.75 g， the metabolite production and the corresponding metabolite production per unit of protein per unit of time were the largest， 1.21 μg and 0.06 μg/[（mg/ml）·h] respectively. Conclusion The optimal condition for preparation of recombinant human CYP2C9 yeast microsomes with minimum content degradation using glass beads lysis process was 30 cycle consist of （30 s per vortex oscillation， 60 s of cooling） and amount of 0.75 g glass beads.
[Key words] Recombinant human CYP2C9 yeast; Microsomes; Glass beads; Diclofenac
基因重組酵母是一种利用基因重组技术，将外源目的基因转录至酵母细胞中，使之表达相应蛋白的酵母[1-3]。重组人代谢酶酵母能够表达人肝微粒中不同亚型的CYP450酶[4]，与其他表达系统（如大肠埃希菌[4-5]、哺乳动物细胞[6]和昆虫细胞[7]等）相比，具有重组酶表达量和催化活性相对较高、生产周期短以及适合于大批量制备药物代谢物等[8]优点，因而具有突出优势。Dragan等[9]通过重组人CYP2C9酵母全细胞反应体系，制备了双氯芬酸的代谢物，但是不同药物的化学结构及理化性质各不相同，因此利用重组人CYP450酵母全细胞反应体系进行不同药物代谢途径及代谢物的筛选仍存在一定困难。
相关热词搜索： 微粒 酵母 重组 法制 破碎上一篇：王者再临为乐而生