摘要
目的:构建和表达人成纤维细胞生长因子-21(FGF21)的基因工程菌,为治疗2型糖尿病的新药开发提供实验数据。方法:通过PCR方法,以质粒pET20b-FGF21为模板扩增FGF21全长,将其与分子伴侣SUMO相融合,再将该融合基因与原核表达载体pET20b连接后转化至BL21(Rosetta)宿主细胞中,通过IPTG诱导获得可溶性表达。将融合蛋白经Ni-NTA、分子筛等进行纯化,获得的蛋白纯度在95%以上。结果:经酶切和基因测序证实获得的FGF21基因序列与GenBank(登录号NM019113)报道的完全一致,构建的pET20b-SUMO-FGF21表达载体在BL21大肠杆菌中成功表达FGF21蛋白,蛋白表达量在15%以上,经SDS-PAGE证实其相对分子质量为19401,与理论预期值相符。Western blotting分析该表达产物与人FGF21多克隆抗体具有特异性结合能力,经SUMO酶酶切后获得纯度大于95%以上的纯蛋白。结论:成功构建表达人FGF21融合蛋白的基因工程菌,经IPTG诱导、Ni-NTA、分子筛等进行纯化后获得了FGF21纯蛋白。
Objective To establish E. colt strain and express human fibroblast growth factor 21 （FGF21）, and provide experimental foundation for development of new drugs for treatment of type 2 diabetes. Methods Human FGF21 gene fragments were obtanined by PCR. After the FGF21 was fused with SUMO （small ubiquitin-related modifier） by PCR, the fused gene was expressed in E. colt BL21 （Rosetta）. By inducing high levels of expression with IPTG, the soluble proteins were obtanined. The fused protein was purified by DEAE Sepharose and Ni-NTA affinity chromatography. Onee cleaved from SUMO, the purity of human FGF21 by SDS-PAGE was shown to be higher than 95%. Results Acquired gene fragments of hFGF21 were identified by digestion and DNA sequencing with the human FGF21 reported it1 GenBank （Accession no. NM019113 ）. The recombinant vector of pET2Ob- SUMO-FGF21 was constructed successfully. SDS-PAGE result proved that SUMO-FGF21 fusion protein with a relative molecular mass of about 19 401 was expressed. Western blotting result showed that the expressed products had specific reaction with anti-human FGF21 polyclonal antibody. Conclusion The engineering E expressing human FGF21 fusion protein is successfully established and the purified protein is obtained.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2010年第1期81-85,共5页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅科技发展计划项目资助课题(20080712
20090249)
吉林省长春净月科技三项费用项目资助课题(2008B003)
