C5补体抑制剂OmCI的重组表达、纯化和活性研究  

Recombinant Expression,Purification and Activity of Complement C5 Inhibitor OmCI

作　　者：汪燕丹 上官雯雯 路建光 黄宗庆 化浩举 蒋宝明 冯军 WANG Yandan;SHANGGUAN Wenwen;LU Jianguang;HUANG Zongqing;HUA Haoju;JIANG Baoming;FENG Jun(Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals,Zhejiang University of Technology,Hangzhou 310014;China State Institute of Pharmaceutical Industry,Shanghai 201203;School of Pharmacy,Fudan University,Shanghai 201203;Shanghai Duomirui Bio-Technology Co.,Ltd.,Shanghai 201203)

机构地区：[1]浙江工业大学长三角绿色制药协同创新中心,浙江杭州310014 [2]中国医药工业研究总院,上海201203 [3]复旦大学药学院,上海201203 [4]上海多米瑞生物技术有限公司,上海201203

出　　处：《中国医药工业杂志》2024年第7期940-947,共8页Chinese Journal of Pharmaceuticals

摘　　要：OmCI(Ornithodoros moubata complement inhibitor,1)是靶向补体C5的药物,用于治疗补体介导的疾病。该研究建立了1的重组、表达、分离、纯化方法,并对其进行活性验证。采用重组大肠埃希菌高密度发酵技术,以胞外可溶形式表达1,融合蛋白的发酵表达产量为317 mg/L。发酵上清经调酸预处理、酶切去除标签蛋白,阴离子交换色谱、反相色谱纯化后得到纯度为98.2%的目的蛋白,总收率为62.8%。目的蛋白经质谱验证,相对分子质量正确;采用体外补体经典途径的抑制试验测得IC50值为8.59 nmol/L,与补体C5的结合常数(KD)为4.74 nmol/L,证明其在大肠埃希菌中成功表达,且具有C5抑制活性。该研究为C5补体抑制剂类药物的高效制备提供了参考。Ornithodoros moubata complement inhibitor(OmCI,1)is a complement C5 inhibitor for the treatment of complement-mediated diseases.In this study,a recombination,expression,isolation and purification method of 1 was established,and activity of the obtained 1 was verified.The protein was expressed in extracellular soluble form by high density fermentation technique of recombinant Escherichia coli(E.coli),and the yield of fusion protein was 317 mg/L.The fermentation supernatant was pretreated by acid-adjusted clarification,enzyme digestion to remove the tagged proteins.Then it was purified by anion-exchange chromatography and reversed-phase chromatography to get the target protein with a purity of 98.2%,and the total yield was 62.8%.The relative molecular weight of target protein was verified to be correct by mass spectrometry.According to the results of the in vitro complement classical pathway inhibition assay,the IC50 and the binding constant(KD)to complement C5 were 8.59 and 4.74 nmol/L,respectively.The results proved that 1 was successfully expressed in E.coli and exhibited C5 inhibitory activity.This study provides a reference for the efficient preparation of complement C5 inhibitors.

关 键 词：C5补体抑制剂(OmCI) 重组表达 发酵 纯化 活性试验 

分 类 号：Q816[生物学—生物工程] Q815