重组人脑利钠肽的纯化与活性测定  

Purification and Activity of Brain Natriuretic Peptide

作　　者：朱重悦[1] 周晓雷[1] 张世光[1] 李海潮[1] 张竞 王岩勃 邹卫[1] 

机构地区：[1]河北科技大学,河北石家庄050018 [2]石家庄沃泰生物科技有限公司,河北石家庄050018

出　　处：《生物技术通讯》2016年第2期223-227,共5页Letters in Biotechnology

基　　金：河北省科技计划(15272401D)

摘　　要：目的:建立重组人脑利钠肽(BNP)的纯化方法,筛选疏水作用层析纯化介质并优化纯化条件。方法:根据带有6×His标签的Dsb A-BNP融合蛋白的特性,采用金属螯合亲和层析(IMAC)、凝血酶酶切去除His-Dsb A、阳离子交换层析等步骤进行粗纯;利用BNP的疏水性,分别选用不同疏水性质的介质Hi Trap Butyl FF、Hi Trap Phenyl(HS)及Source 15进行精纯;用SDS-PAGE和HPLC检测纯化获得的BNP的含量及纯度;用细胞法对BNP进行活性检测。结果:经IMAC、酶切去除标签蛋白及阳离子交换层析纯化后,获得纯度为60%的BNP;Hi Trap Butyl FF、Hi Trap Phenyl(HS)等疏水介质与BNP的吸附效果不佳,而经Source 15反相层析后,BNP的纯度可达98.98%,活性检测与对照品一致。结论:利用Source 15反相层析可获得高纯度、活性优的BNP原液,可满足后期制剂处方实验的要求,为后续研究奠定了基础。Objective： To develop a purification procedure of human brain natriuretic peptide（BNP）, and screenthe media of hydrophobic interaction in purification. Methods： According to the characteristics of Dsb A- BNP,BNP was preliminary purified by the procedure, consisting of IMAC, enzyme digestion and ion exchange chromatog-raphy. According to the hydrophobic of BNP, choose different hydrophobic medium, including Hi Trap Butyl FF, Hi-Trap Phenyl（HS） and Source 15, for polishing purification. The purified protein was determined for content andpurity of target protein by SDS-PAGE and HPLC, and analyzed for protein activity by cell test. Results： The puri-ty of BNP was more than 60% after preliminary purification. The purification effect of RPLC was better than HIC.The purity of targe protein reached 98.98%, and the protein activity agreed with reference standard substance.Conclusion： The BNP can be used for the study of preparation after being purified by reverse phase high perfor-mance liquid chromatography with Source 15 medium.

关 键 词：人脑利钠肽 疏水作用 蛋白活性 

分 类 号：Q502[生物学—生物化学]