摘要
建立从猪眼晶状体中提取和一次过柱分离纯化波形蛋白(Vim)的工艺方法。以精提液溶解猪眼晶状体粗提粉,使用单因素实验及响应面分析对精提温度、时间、料液比、p H值四个因素进行优化,利用DEAE Sepharose FF阴离子交换柱分离纯化目标蛋白,以免疫印迹法鉴定波形蛋白,10%SDS-PAGE检测纯化Vim的纯度和相对回收率。精提的最佳工艺条件为:温度13.16℃,时间26.01 min,料液比1∶34(g∶m L),p H7.03。使用DEAE Sepharose FF柱以不少于40倍柱体积的36 mmol/L Na H2PO4缓冲液洗脱后,以100倍柱体积的36—86 mmol/L Na H2PO4缓冲液一次线性洗脱分离Vim。经10%SDS-PAGE结合Image Lab 4.0分析软件检测纯化制得的冻干粉中Vim蛋白纯度达(80.66±1.30)%,相对回收率为(36.56±0.96)%。
This study is aimed to establish a purifying method of vimentin（ Vim） extracted from porcine lens by once separation on column. The crude extracts of porcine lens were dissolved by refined extracting solution. The temperature,time,solid to liquid ratio and solution p H of refined extraction were optimized using single factor experiment and response surface analysis. Target protein was isolated and purified via DEAE Sepharose fast flow anion exchange column and Vim was identified by Western-Blot. The 10% SDS-PAGE was used to detect the purity and relative recovery rate. The optimized temperature,time,solid to liquid ratio and solution p H of refined extraction respectively were 13. 16 ℃,26. 01 min,1： 34（ g： m L） and p H7. 03. Followed elution with 36 mmol / L Na H2PO4 buffer of 40 times volume of the column,36-86 mmol / L Na H2PO4 buffer of 100 times volume of the column was used to linear elution once for isolating and purifying Vim. Analyzing the electrophoretogram of 10% SDS-PAGE with Image Lab 4. 0 software,the purity of Vim in purified lyophilized powder was（ 80. 66 ± 1. 30） % and the relative recovery rate was（ 36. 56 ± 0. 96） %.
出处
《武汉轻工大学学报》
2016年第3期29-34,共6页
Journal of Wuhan Polytechnic University
基金
湖北省自然科学基金(2015CFC845)
武汉轻工大学研究生创新项目(2013cx021)
关键词
猪
波形蛋白
提取
纯化
pig
vimentin
extraction
purification
