重组圆锥芋螺胰岛素G1的原核表达与降糖活性检测

Prokaryotic expression and hypoglycemic activity determination of insulin G1 from Conus geographus

摘要:

为缩短重组胰岛素起效时间，达到快速降低餐后血糖的作用，以圆锥芋螺胰岛素G1 (cI G1) 为研究对象，参照人胰岛素原 (hPI) 和圆锥芋螺胰岛素G1原 (cPI G1) 基因设计重组cPI G1的核苷酸序列，按照大肠杆菌Escherichia coli密码子使用频率进行密码子优化后构建pET22b(+)-cPI G1质粒，以E. coli BL21(DE3) 菌株为宿主进行原核表达，获得重组cPI G1蛋白，经胰蛋白酶切割及纯化得到重组cI G1，其效价为25.9 IU/mg。空腹血糖测试 (FBGT) 和葡萄糖耐量实验 (OGTT) 表明，cI G1可迅速降低正常和链脲佐菌素 (STZ) 致糖尿病模型小鼠的血糖，但持续时间短，研究结果为重组速效胰岛素的开发提供参考。

Abstract:

Rapid reduction of postprandial blood glucose is very beneficial to diabetics. In order to shorten the onset time of recombinant insulin, the cone snail insulin G1 (cI G1) of Conus geographus was studied. First, the nucleotide sequence of recombinant cone snail proinsulin G1 (cPI G1) was designed and synthesized according to the genes of human proinsulin (hPI) and cPI G1. The codon was optimized according to Escherichia coli (E. coli) codon usage frequency. Then, the plasmid pET22b(+)-cPI G1 was constructed and the recombinant cPI G1 was expressed in E. coli BL21(DE3) host strain. The recombinant cPI G1 was then purified and cleaved specially by trypsin to generate the recombinant cI G1, and its potency is 25.9 IU/mg. Fasting blood glucose test (FBGT) and oral glucose tolerance test (OGTT) suggested that the recombinant cI G1 could rapidly reduce blood glucose in normal and streptozotocin (STZ)-induced diabetic mice, but only for a short duration. This study provides a technical reference for the development of recombinant fast-acting insulin.