人FGF-21基因的克隆、表达及其调节脂肪细胞糖代谢的活性
收稿日期: 2009-10-19
修回日期: 2010-04-13
网络出版日期: 2010-05-24
基金资助
黑龙江省科技厅重点攻关项目(编号：2006G0461-00)、黑龙江省博士后科研启动资助金、黑龙江省教育厅项目(编号：11521022)和哈尔滨市科技创新人才研究专项资金(编号：2008RFQXS017)项目
Cloning, expression and glucose regulation activity of human FGF-21
Received date: 2009-10-19
Revised date: 2010-04-13
Online published: 2010-05-24
成纤维细胞生长因子FGF-21是FGF家族的一个新成员, 最近的研究发现其具有调节血糖的作用, 有望成为治疗2型糖尿病的基因药物。文章应用RT-PCR技术, 从成人肝脏中克隆人源的FGF-21成熟蛋白基因, 并将其克隆到T载体上, 经测序鉴定后, 将其亚克隆到原核表达载体pSUMO上, 转入大肠杆菌Rosetta(DE3)中。鉴定阳性克隆后, 用IPTG诱导FGF-21表达, 并用Ni-NTA柱进行亲和层析纯化。以3T3-L1脂肪细胞的葡萄糖吸收实验来鉴定FGF-21表达产物促进糖吸收的活性。结果表明, FGF-21成熟蛋白基因大小为546 bp, 测序结果与GenBank数据库中的序列一致。SDS-PAGE与Western blotting结果表明: 人源FGF-21成熟蛋白大小19.4 kDa, 经3T3-L1脂肪细胞的葡萄糖吸收实验验证其具有促进葡萄糖吸收的生物活性, 并且GLUT1是FGF-21发挥生物学作用的终末执行单位。
关键词： 人源FGF-21; 成熟蛋白; 糖尿病; 小泛素相关修饰蛋白表达载体
侯玉婷，李晋南，任桂萍，刘铭瑶，孙国鹏，王文飞，李德山 . 人FGF-21基因的克隆、表达及其调节脂肪细胞糖代谢的活性[J]. 遗传, 2010 , 32(6) : 583 -587 . DOI: 10.3724/SP.J.1005.2010.00583
Fibroblast growth factor (FGF)-21 is a recently discovered glucose regulator and has potential to become therapeutics for treatment of type 2 diabetes. The aim of this study was to clone and express human FGF-21 gene and characterize its bioactivity for glucose regulation. The hFGF-21 cDNA was cloned from human liver by RT-PCR and subcloned into the pSUMO vector after sequencing confirmation. The recombinant plasmid was transformed into Escherichia coli Rosetta strain. The FGF-21 protein expression was induced by IPTG and purified by Ni-NTA agarose. The FGF-21 product was verified by Western blotting analysis with specific antibody. The bioactivity of the purified protein was examined by glucose uptake assay in 3T3-L1 adipocytes. The cloned hFGF-21 gene consisted of 546 bp, which was in agreement with the published data in GenBank. SDS-PAGE analysis showed that hFGF-21 expressed in the E. coli system was 19.4 kDa in size. The glucose uptake assay in 3T3-L1 adipocytes indicated that the purified hFGF-21 could stimulate glucose uptake in a dose-dependent manner, and glucose transporters (GLUT1) is the functional unit.
Key words： human FGF-21; mature protein; diabetes mellitus; SUMO
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