摘要
为了实现人源化胰岛素样生长因子Ⅰ(human insulin-like growth factors-Ⅰ,hIGF-Ⅰ)基因在大肠杆菌中的高效表达,了解IGF-Ⅰ基因结构与功能的关系,探索人工制备的hIGF-Ⅰ在临床治疗疾病上的应用,实验参照GenBank中人IGF-Ⅰ氨基酸序列并进行密码子优化,然后人工合成这条基因并将克隆至pUC57载体上.构建重组表达载体pGEX-6P-1-hIGF-Ⅰ,转化大肠杆菌BL21菌株,进行不同诱导条件的优化,SDS-PAGE电泳确定最佳诱导条件,并利用建立hIGF-Ⅰ的间接ELISA检测方法分析表达产物活性.结果表明,获得重组表达载体pGEX-6P-1-hIGF-Ⅰ,并在大肠杆菌中实现与GST蛋白的融合可溶性表达.最佳诱导条件OD600值为0.8、IPTG浓度为0.5 mmol/L,表达蛋白的浓度为2.811 mg/mL,且产物具有抗原活性.说明人工制备的hIGF-Ⅰ与天然的hIGF-Ⅰ生物活性有部分相同,在基础研究和药物开发方面可作为替代物进行研究.
ObjectivesConstructing a fused expression vector of synthesizing human long-chain IGF-Ⅰgene and efficiently expressed it in E.coli,in order to lay the foundation for the development of synthetic human IGF-Ⅰ which can be used to therapy various diseases in clinical treatment.Methods Reference amino acid sequence of human IGF-Ⅰin Genbank and optimize the codon,synthesized this gene then linked into the pUC57 cloning vector,enzyme cleavaged the plasmid and then gel extraction to get the fragment.The fragment was cloned into the pGEX-6 P-1 vector to get the recombinant expression vector pGEX-6 P-1-hIGF-Ⅰ,transformed into BL21,induced by IPTG,SDS-PAGE electrophoresis analysis of expression products,and use ELISA to detect the activity of expressed protein.Results Obtained with recombinant hIGF-Ⅰ expression vector and successfully expressed in E.coli.ConclusionsThis experiment lays the theoretical foundation for the analysis of the relationship between its IGF-Ⅰ genetic structure and function,and provides basis for further basic research and development of clinical application prospect drugs.
作者
李向茸
王兴陇
李倩
邓盈盈
梁浩勤
周飚
冯若飞
LI Xiang-rong;WANG Xing-long;LI Qian;DENG Ying-ying;LIANG Hao-qin;ZHOU Biao;FENG Ruo-fei(The Key Bio-engineering and Technology Laboratory of NationalityCommission,Northwest Minzu University,Lanzhou 730030,China;Gansu Engineering Research Center for Animal Cell,Lanzhou 730030,China;Life Science and Engineering College of Northwest Minzu University,Lanzhou 730030,China)
出处
《西北民族大学学报（自然科学版）》
2018年第1期20-25,共6页
Journal of Northwest Minzu University(Natural Science)
基金
教育部"长江学者和创新团队发展计划"项目(IRT_17R88)
中央高校基本科研业务费专项研究生科研创新项目(Yxm2017119)
