人成纤维细胞生长因子-21突变体的制备及活性探究  

Preparation and Activity Analysis of Mutant Human Fibrablast Growth Factor 21 Protein

作　　者：刘全文 唐诗洁 郭振昌 胡昌华[1] 刘雪梅[1] LIU Quan-wen;TANG Shi-jie;GUO Zhen-chang;HU Chang-hua;LIU Xue-mei(Southwest University College of Pharmaceutical Sciences,Chongqing 400715,China)

机构地区：[1]西南大学药学院,重庆400715

出　　处：《药物生物技术》2023年第4期342-347,共6页Pharmaceutical Biotechnology

基　　金：2020重庆市自然科学基金项目(No.cstc2020jcyj⁃msxmX0424);重庆市留学人员回国创业创新支持计划项目(No.cx2018089)。

摘　　要：成纤维细胞生长因子(Fibrablast growth factors,FGFs)是一组具有保守核心结构域(约120个氨基酸)的信号蛋白,包括6个亚家族成员。FGF21是FGF19亚家族中的一员,作为重要的代谢调控信号分子,FGF21作用于肝脏、胰岛和脂肪组织,在肥胖、非酒精性脂肪肝、Ⅱ型糖尿病等疾病的临床诊疗中极具潜力。研究发现人FGF21(hFGF21)在大肠杆菌中表达量低、半衰期短、稳定性差,严重制约其成药可能性。本研究通过生物学信息分析,利用密码子优化策略调整密码子偏好性,以pET-32a(+)-Fgf21表达载体为模板,采用定点突变,构建突变载体,在大肠杆菌中进行表达并纯化。结果显示,将FGF21的58位亮氨酸变为缬氨酸(L58V),以及108位谷氨酰胺变为精氨酸(Q108R),突变蛋白(mutFGF21((L58V)),mutFGF21((Q108R)))在大肠杆菌中可实现高效可溶表达。HepG2细胞糖吸收试验进一步显示,mutFGF21((L58V))和mutFGF21((Q108R))蛋白处理后的细胞对葡萄糖的摄取利用增加,培养基中残余葡萄糖量减少,与野生型相比,突变型蛋白促进糖吸收效果随着蛋白浓度增加更为明显。FGFs(Fibrablast growth factors)is a group of signaling proteins with a conserved core domain(about 120 amino acids),including six subfamily members.FGF21 is a member of the FGF19 subfamily.As an important signaling molecule of meta⁃bolic regulation,FGF21 acts on liver,islet and adipose tissue.FGF21 has great potential in the clinical diagnosis and treatment of obesity,non⁃alcoholic fatty liver disease,type II diabetes and other diseases.It was found that human FGF21(hFGF21)had low expression level,short half⁃life and poor stability in Escherichia coli,which seriously restricted the availability of drugs.To overcome the problem,codon optimization strategy was used to adjust codon preference through biological information analysis.Using the expression vector PET⁃32A(+)⁃FGF21 as template,site⁃directed mutation was adopted to construct mutant vector,which was expressed and purified in E.coli.The results showed that mutFGF21 protein has good solubility in E.coli by changing the 58th L into V(L58V)or the 108th Q into R(Q108R).HepG2 cell glucose absorption assay further showed that treated cells with mutFGF21 protein increased glucose uptake and utilization,and reduced the amount of residual glucose in the culture medium.The effect of the mutant protein on glucose absorption was more obvious with the increase of protein concentration compared with the wild⁃type protein.

关 键 词：FGF21 序列分析 密码子优化 蛋白表达 定点突变 活性研究 

分 类 号：Q51[生物学—生物化学]