摘要
利用SUMO表达载体在大肠杆菌中高效可溶性表达鼠源成纤维细胞生长因子(FGF-21)成熟蛋白,并研究其调节血糖的功能。将鼠源FGF-21成熟蛋白基因亚克隆至SUMO表达载体上,在大肠杆菌Rosetta(DE3)中诱导表达,并用镍离子螯合柱(Ni-NTA)纯化重组蛋白,透析后利用SUMO蛋白酶I切割融合蛋白,获得纯度较高的成熟蛋白。将3T3-L1成纤维细胞分化成脂肪细胞,经微量化的GOD-POD法检测培养基中葡萄糖含量,统计学分析葡萄糖消耗率。结果表明,在SUMO表达体系中鼠源FGF-21成熟蛋白基因以可溶形式表达,且可被SUMO蛋白酶I有效地切割,获得纯度较高的成熟蛋白。与未经处理的脂肪细胞对照组相比,经鼠源FGF-21成熟蛋白处理后脂肪细胞对葡萄糖的摄取利用显著增加,残存在培养基中的葡萄糖含量明显减少;两样本比较的t检验,P<0.001,差异极显著。
The objective of the paper is to utilize SUMO expression vector to efficiently express mouse fibroblast growth factor （FGF）-21. Glucose regulation activity was studied for mouse mature FGF-21 protein. The cDNA of mouse mature FGF-21 protein was subcloned into the SUMO expression vector. The recombinant plasmids were transformed into Escherichia coli Rosetta （DE3） and the expressed recombinant protein was purified by its binding column. The purified recombinant protein was dialyzed against PBS. The fusion protein could be efficiently cleaved by SUMO protease I to obtain pure and active recombinant protein. To examine the glucose regulation activity of mFGF-21, 3T3-L1 pre-adipocytes were differentiated into adipocytes, glucose uptake activity of mFGF-21 was examined by Glucose Oxidase and Peroxidase （GOD-POD） assay. The cDNA of mouse mature FGF-21 protein was expressed in soluble form and the recombinant protein accounted for 50% of total bacterial protein in the SUMO expression system. Mouse FGF-21 possessed strong bioactivity for glucose homeostasis in 3T3-L1 adipocytes. And mouse FGF-21 could stimulate glucose uptake of 3T3-L1 adipocytes in dose-dependent manner.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2009年第5期62-67,共6页
Journal of Northeast Agricultural University
基金
黑龙江省科技厅重点攻关项目(2006G0461-00)
