摘要
促红细胞生成素（EPO）是一种多效细胞因子,具有促进红细胞生成和组织修复作用,在用于组织修复时会导致血粘度升高,改变其结构降低副作用是其应用的关键。我们对EPO进行定点突变,筛选能够降低促红细胞生成的突变体,为进一步组织修复研究提供基础。本实验中我们利用定点突变试剂盒分别将人源性促红细胞生成素中7个赖氨酸突变为谷氨酰胺;原核表达并纯化突变蛋白;所得产物腹腔注射KM小鼠体内,通过检测小鼠血中的网织红细胞指数考察mEPO（mutantEPO）的活性。rhEPO（recombinantEPO）及7个mEPO组的网织红细胞指数与阴性对照组比较,p值均小于0.05,有显著性差异;mEPO（K97Q）与rhEPO阳性对照组比较,p〈0.05有显著性差异,这说明原核表达的rhEPO及mEPO均具有促红细胞生成活性,mEPO（K97Q）的促红细胞生成作用明显降低,可能在组织修复过程中减少副作用的发生,为进一步多点突变奠定了基础。
Erythropoietin （EPO） is a muti-effect cytokines. It causes higher blood viscosity when used for tissue repair, so the key to its application is changing its structure to reduce the side effects. We fixed point mutation of EPO that could be chosen to reduce erythropoiesis and lay a foundation for the further reseach for tissue repair. We used the product kits to mutate 7 lysine ofrhEPO （reconbinant EPO） into glutamine; we prokaryotic expressed and purified the mutant proteins. We detected the reticulocyte indices of rats to investigate the activity of mEPO （mutant EPO） after the rats were intraperoneal injected into mutant protein and p 〈0.05 when the reticulocyte indices of rhEPO and seven mEPO group compared with negative control group, which shows there are significant differences, p〈0.05 when mEPO （K97Q） compared with rhEPO positive control group which shows there are significant differences. Both the prokaryotic expression of rhEPO and mEPO have erythropoiesis .The effect of mEPO （K97Q） on promoting erythropoiesis is decreased obviously which may reduce the occurrence of side effects in the process of tissue repair. We provided the foundations for the further multipoint mutation.
作者
赵佳浩
高秀峰
潘佳欣
罗亚雄
吴晓婷
李永生
Zhao Jiahao;Gao Xiufeng;Pan Jiaxin;Luo Yaxiong;Wu Xiaoting;Li Yongsheng(West China School of Preclinical and Forensic Medicine, Sichuan University, Sichuan, 610041;Department of Chemical Analysis School of Chemical Engineering, Sichuan University, Sichuan, 610065)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2018年第7期3125-3131,共7页
Genomics and Applied Biology
基金
四川大学化学与工程学院的支持
关键词
促红细胞生成素
原核表达
分离纯化
体内活性检测
定点突变
Erythropoietin
Prokaryotic expression
Separation and purification
Detection activity in vivo
Site-directed mutagenesis
