重组圆锥芋螺胰岛素G1的原核表达与降糖活性检测
|
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。
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 pET22 b(+)-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.
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 pET22 b(+)-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.
引文
[1]Lu P,Shen L.Thirty years of experience with recombinant human insulin-from familiarity to reliance.Chin J Diabetes,2018,26(1):84-88(in Chinese).鲁平,沈丽.重组人胰岛素30载.中国糖尿病杂志,2018,26(1):84-88.
[2]Gast K,Schüler A,Wolff M,et al.Rapid-acting and human insulins:hexamer dissociation kinetics upon dilution of the pharmaceutical formulation.Pharm Res,2017,34(11):2270-2286.
[3]Mbanya JC,Sandow J,Landgraf W,et al.Recombinant human insulin in global diabetes management-focus on clinical efficacy.Eur Endocrinol,2017,13(1):21-25.
[4]Bagchi K,Roy S.Sensitivity of water dynamics to biologically significant surfaces of monomeric insulin:role of topology and electrostatic interactions.J Phys Chem B,2014,118(14):3805-3813.
[5]Menting JG,Gajewiak J,MacRaild CA,et al.Aminimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin.Nat Struct Mol Biol,2016,23(10):916-920.
[6]Sch?gger H.Tricine-SDS-PAGE.Nat Protoc,2006,1(1):16-22.
[7]Castellanos-Serra LR,Hardy E,Ubieta R,et al.Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence.FEBS Lett,1996,378(2):171-176.
[8]National Pharmacopoeia Committee.Pharmacopoeia of Peoples Republic of China.Part 2.Beijing:China Medical Science Press,2015:789-790(in Chinese).国家药典委员会.中华人民共和国药典.二部.北京:中国医药科技出版社,2015:789-790.
[9]Zheng GJ,Ni L,Guo DJ.Expression of human insulin precursor in Pichia pastoris using GAPpromoter and activity of expressed product.Chin JBiol,2013,26(12):1753-1757,1763(in Chinese).郑国君,倪玲,郭德军.人胰岛素前体在毕赤酵母中的组成型表达及其活性.中国生物制品学杂志,2013,26(12):1753-1757,1763.
[10]Elsayed AM,Khaled AH,Al Remawi MM,et al.Low molecular weight chitosan-insulin complexes solubilized in a mixture of self-assembled labrosol and plurol oleaque and their glucose reduction activity in rats.Mar Drugs,2018,16(1):32-47.
[11]Wu RS,Fan SJ,Mei Y,et al.The effect of blood sugar level after insulin skin-poping in mice.Prog Mod Biomed,2012,12(5):852-855(in Chinese).吴润生,范世锦,梅赟,等.胰岛素小鼠皮下注射的降血糖作用研究.现代生物医学进展,2012,12(5):852-855.
[12]Li N,Yang ZC,Jin JY,et al.Hypoglycemic effect of Long Hu Ren Dan on STZ-induced diabetic mice.JGuangdong Pharm Univ,2018,34(1):69-72(in Chinese).李柠,杨智承,金家骅,等.龙虎人丹对链脲佐菌素诱导糖尿病小鼠降糖作用研究.广东药科大学学报,2018,34(1):69-72.
[13]Racsa PN,Meah Y,Ellis JJ,et al.Comparative effectiveness of rapid-acting insulins in adults with diabetes.J Manag Care Spec Pharm,2017,23(3):291-298.
[14]Duckworth WC,Bennett RG,Hamel FG.Insulin degradation:progress and potential.Endocr Rev,1998,19(5):608-624.
[15]Tang WJ.Targeting insulin-degrading enzyme to treat type 2 diabetes mellitus.Trends Endocrinol Metab,2016,27(1):24-34.
[16]Li JD,Wang H.Strategies to improve the folding and modification of recombinant proteins:a review.Chin J Biotech,2017,33(4):591-600(in Chinese).李家冬,王弘.重组蛋白正确折叠与修饰的提高策略.生物工程学报,2017,33(4):591-600.
[2]Gast K,Schüler A,Wolff M,et al.Rapid-acting and human insulins:hexamer dissociation kinetics upon dilution of the pharmaceutical formulation.Pharm Res,2017,34(11):2270-2286.
[3]Mbanya JC,Sandow J,Landgraf W,et al.Recombinant human insulin in global diabetes management-focus on clinical efficacy.Eur Endocrinol,2017,13(1):21-25.
[4]Bagchi K,Roy S.Sensitivity of water dynamics to biologically significant surfaces of monomeric insulin:role of topology and electrostatic interactions.J Phys Chem B,2014,118(14):3805-3813.
[5]Menting JG,Gajewiak J,MacRaild CA,et al.Aminimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin.Nat Struct Mol Biol,2016,23(10):916-920.
[6]Sch?gger H.Tricine-SDS-PAGE.Nat Protoc,2006,1(1):16-22.
[7]Castellanos-Serra LR,Hardy E,Ubieta R,et al.Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence.FEBS Lett,1996,378(2):171-176.
[8]National Pharmacopoeia Committee.Pharmacopoeia of Peoples Republic of China.Part 2.Beijing:China Medical Science Press,2015:789-790(in Chinese).国家药典委员会.中华人民共和国药典.二部.北京:中国医药科技出版社,2015:789-790.
[9]Zheng GJ,Ni L,Guo DJ.Expression of human insulin precursor in Pichia pastoris using GAPpromoter and activity of expressed product.Chin JBiol,2013,26(12):1753-1757,1763(in Chinese).郑国君,倪玲,郭德军.人胰岛素前体在毕赤酵母中的组成型表达及其活性.中国生物制品学杂志,2013,26(12):1753-1757,1763.
[10]Elsayed AM,Khaled AH,Al Remawi MM,et al.Low molecular weight chitosan-insulin complexes solubilized in a mixture of self-assembled labrosol and plurol oleaque and their glucose reduction activity in rats.Mar Drugs,2018,16(1):32-47.
[11]Wu RS,Fan SJ,Mei Y,et al.The effect of blood sugar level after insulin skin-poping in mice.Prog Mod Biomed,2012,12(5):852-855(in Chinese).吴润生,范世锦,梅赟,等.胰岛素小鼠皮下注射的降血糖作用研究.现代生物医学进展,2012,12(5):852-855.
[12]Li N,Yang ZC,Jin JY,et al.Hypoglycemic effect of Long Hu Ren Dan on STZ-induced diabetic mice.JGuangdong Pharm Univ,2018,34(1):69-72(in Chinese).李柠,杨智承,金家骅,等.龙虎人丹对链脲佐菌素诱导糖尿病小鼠降糖作用研究.广东药科大学学报,2018,34(1):69-72.
[13]Racsa PN,Meah Y,Ellis JJ,et al.Comparative effectiveness of rapid-acting insulins in adults with diabetes.J Manag Care Spec Pharm,2017,23(3):291-298.
[14]Duckworth WC,Bennett RG,Hamel FG.Insulin degradation:progress and potential.Endocr Rev,1998,19(5):608-624.
[15]Tang WJ.Targeting insulin-degrading enzyme to treat type 2 diabetes mellitus.Trends Endocrinol Metab,2016,27(1):24-34.
[16]Li JD,Wang H.Strategies to improve the folding and modification of recombinant proteins:a review.Chin J Biotech,2017,33(4):591-600(in Chinese).李家冬,王弘.重组蛋白正确折叠与修饰的提高策略.生物工程学报,2017,33(4):591-600.