SUMO-cFGF-21发酵工艺优化及其降糖效果研究
|
摘要
研究旨在优化SUMO-cFGF-21发酵工艺,同时检测cFGF-21蛋白对糖尿病小鼠和犬血糖的降糖效果。通过SDS-PAGE电泳分析发酵条件(菌种、工程菌OD600、诱导时间、诱导温度、诱导剂IPTG浓度)对SUMOcFGF-21蛋白表达的影响。结果表明,22号菌种蛋白表达量最高,达到上清蛋白30.5%,可作为一级菌种。发酵过程中,当工程菌OD600值约为2、诱导时间6 h、诱导温度23℃、诱导剂IPTG终浓度1 mmol·L~(-1)时,上清中SUMOcFGF-21蛋白表达量最高,达到上清蛋白19.7%。经Ni柱亲和层析,酶切去掉SUMO标签后成功制备cFGF-21蛋白。将不同剂量cFGF-21持续注射糖尿病小鼠两周,以有效剂量cFGF-21持续注射糖尿病犬两周,观察降糖效果。结果表明,与模型对照组相比,有效剂量cFGF-21均可极显著降低糖尿病小鼠和犬的血糖(P<0.01)。研究成功优化SUMO-cFGF-21蛋白中试发酵工艺,并检测cFGF-21蛋白对糖尿病小鼠和犬血糖的降糖效果,为临床应用cFGF-21治疗犬糖尿病提供依据。
The aim of this study was to optimize the fermentation process of SUMO-cFGF-21 and to detect the hypoglycemic effect of cFGF-21 protein on diabetic mice and canine. The effects of fermentation conditions(bacteria, the OD600 of engineered bacteria, induction time, induction temperature and the concentration of IPTG) on SUMO-cFGF-21 protein expression were analyzed by SDS-PAGE electrophoresis. The results showed that the protein expression of number 22 was the highest, reaching 30.5% of the supernatant protein, as the original bacteria. During the fermentation process, when the OD600 of engineering bacteria reached about 2, induction time was 6 h, induction temperature was 23 ℃, the final concentration of IPTG was 1 mmol·L~(-1), the expression of SUMO-cFGF-21 protein in supernatant was the highest, and up to 19.7%. cFGF-21 protein without the SUMO tag was successfully prepared by Ni column affinity chromatography. Different doses of cFGF-21 were administered into diabetic mice for two weeks, an effective dose of c FGF-21 was administered to diabetic canine for two weeks to observe the hypoglycemic effect. The results showed that compared with the model control group, effective dose of cFGF-21 could significantly reduce blood glucose in diabetic mice and canine(P<0.01). The fermentation process of SUMO-c FGF-21 was successfully optimized in the study, and the hypoglycemic effect of cFGF-21 protein on diabetic mice and canine was detected. It provides the basis for the clinical application of cFGF-21 in the treatment of canine diabetes.
The aim of this study was to optimize the fermentation process of SUMO-cFGF-21 and to detect the hypoglycemic effect of cFGF-21 protein on diabetic mice and canine. The effects of fermentation conditions(bacteria, the OD600 of engineered bacteria, induction time, induction temperature and the concentration of IPTG) on SUMO-cFGF-21 protein expression were analyzed by SDS-PAGE electrophoresis. The results showed that the protein expression of number 22 was the highest, reaching 30.5% of the supernatant protein, as the original bacteria. During the fermentation process, when the OD600 of engineering bacteria reached about 2, induction time was 6 h, induction temperature was 23 ℃, the final concentration of IPTG was 1 mmol·L~(-1), the expression of SUMO-cFGF-21 protein in supernatant was the highest, and up to 19.7%. cFGF-21 protein without the SUMO tag was successfully prepared by Ni column affinity chromatography. Different doses of cFGF-21 were administered into diabetic mice for two weeks, an effective dose of c FGF-21 was administered to diabetic canine for two weeks to observe the hypoglycemic effect. The results showed that compared with the model control group, effective dose of cFGF-21 could significantly reduce blood glucose in diabetic mice and canine(P<0.01). The fermentation process of SUMO-c FGF-21 was successfully optimized in the study, and the hypoglycemic effect of cFGF-21 protein on diabetic mice and canine was detected. It provides the basis for the clinical application of cFGF-21 in the treatment of canine diabetes.
引文
[1]Batchelor D J,German A J,Shirazi-Beechey S P.Relevance of sodium/glucose cotransporter-1(SGLT1)to diabetes mellitus and obesity in dogs[J].Domestic Animal Endocrinology,2013,44(3):139-144.
[2]Adams J P,Holder A L,Catchpole B.Recombinant canine single chain insulin analogues:Insulin receptor binding capacity and ability to stimulate glucose uptake[J].Veterinary Journal,2014,202(3):436-442.
[3]杜宏超,庞海东,林德贵.犬糖尿病胰岛素疗法[J].中国兽医杂志,2017(3):111-114.
[4]贺星亮,曲志学.如何诊治犬的糖尿病[J].中国工作犬业,2015(1):22-23.
[5]Hoenig M.Comparative aspects of diabetes mellitus in dogs and cats[J].Molecular and Cellular Endocrinology,2002,197(1-2):221-229.
[6]Mattin M,O'Neill D,Church D,et al.An epidemiological study of diabetes mellitus in dogs attending first opinion practice in the UK[J].Veterinary Record,2014,174(14):349.
[7]Xu P,Zhang Y,Jiang X,et al.Canine fibroblast growth factor 21ameliorates hyperglycemia associated with inhibiting hepatic gluconeogenesis and improving pancreatic beta-cell survival in diabetic mice and dogs[J].Plos One,2016,11(5):e0155598.
[8]Itoh N,Ornitz D M.Functional evolutionary history of the mouse Fgf gene family[J].Dev Dyn,2010,237(1):18-27.
[9]杨文琦,凌文华.成纤维细胞生长因子21对糖脂代谢调控的研究进展[J].生理科学进展,2016,47(4):260-264.
[10]雷营,祁范范,周亚茹.成纤维细胞生长因子21对代谢性疾病影响的研究进展[J].中国临床医生杂志,2016,44(10):14-16.
[11]任桂萍,李璐,孙国鹏,等.成纤维细胞生长因子(FGF)受体-2参与FGF-21介导的糖代谢活性[J].生物化学与生物物理进展,2009,36(2):165-174.
[12]Coskun T,Bina H A,Schneider M A,et al.Fibroblast growth factor 21 corrects obesity in mice[J].Endocrinology,2008,149(12):6018-6027.
[13]Chen Y,Wang J J,Li J,et al.Activating transcription factor 4mediates hyperglycaemia-induced endothelial inflammation and retinal vascular leakage through activation of STAT3 in a mouse model of type 1 diabetes[J].Diabetologia,2012,55(9):2533-2545.
[14]李德山,姜兴昊,徐鹏飞,等.不同维持剂量聚乙二醇修饰的FGF-21在Ⅰ型糖尿病小鼠中降糖作用的研究[J].东北农业大学学报,2017(2):69-75.
[15]许会静,刘磊,董媛,等.FGF21对糖尿病Beagle犬降糖作用的研究[J].生物技术,2016(2):188-192.
[16]Xue C,Zhao J,Chen L,et al.Recent advances and state-of-theart strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum[J].Biotechnology Advances,2017,35(2):310-322.
[17]周海岩,刘龙,堵国成,等.诱导温度对大肠杆菌BR-42ΔpykA(p AP-B03)发酵生产L-苯丙氨酸的影响[J].食品与发酵工业,2014,40(4):7-12.
[18]Xu P,Ye X,Zhang Y,et al.Long-acting hypoglycemic effects of PEGylated FGF21 and insulin glargine in mice with type 1diabetes[J].Journal of Diabetes and its Complications,2015,29(1):5-12.
[19]何昆,张雅坤,叶贤龙,等.精氨酸修饰成纤维细胞生长因子21及修饰后蛋白体内稳定性和糖代谢调节作用的研究[J].生物化学与生物物理进展,2012,39(11):1089-1098.
[20]Thombre S,Gadgil M.Increase in efficiency of media utilization for recombinant protein production in Chinese hamster ovary culture through dilution[J].Biotechnology and Applied Biochemistry,2011,58(1):25-31.
[2]Adams J P,Holder A L,Catchpole B.Recombinant canine single chain insulin analogues:Insulin receptor binding capacity and ability to stimulate glucose uptake[J].Veterinary Journal,2014,202(3):436-442.
[3]杜宏超,庞海东,林德贵.犬糖尿病胰岛素疗法[J].中国兽医杂志,2017(3):111-114.
[4]贺星亮,曲志学.如何诊治犬的糖尿病[J].中国工作犬业,2015(1):22-23.
[5]Hoenig M.Comparative aspects of diabetes mellitus in dogs and cats[J].Molecular and Cellular Endocrinology,2002,197(1-2):221-229.
[6]Mattin M,O'Neill D,Church D,et al.An epidemiological study of diabetes mellitus in dogs attending first opinion practice in the UK[J].Veterinary Record,2014,174(14):349.
[7]Xu P,Zhang Y,Jiang X,et al.Canine fibroblast growth factor 21ameliorates hyperglycemia associated with inhibiting hepatic gluconeogenesis and improving pancreatic beta-cell survival in diabetic mice and dogs[J].Plos One,2016,11(5):e0155598.
[8]Itoh N,Ornitz D M.Functional evolutionary history of the mouse Fgf gene family[J].Dev Dyn,2010,237(1):18-27.
[9]杨文琦,凌文华.成纤维细胞生长因子21对糖脂代谢调控的研究进展[J].生理科学进展,2016,47(4):260-264.
[10]雷营,祁范范,周亚茹.成纤维细胞生长因子21对代谢性疾病影响的研究进展[J].中国临床医生杂志,2016,44(10):14-16.
[11]任桂萍,李璐,孙国鹏,等.成纤维细胞生长因子(FGF)受体-2参与FGF-21介导的糖代谢活性[J].生物化学与生物物理进展,2009,36(2):165-174.
[12]Coskun T,Bina H A,Schneider M A,et al.Fibroblast growth factor 21 corrects obesity in mice[J].Endocrinology,2008,149(12):6018-6027.
[13]Chen Y,Wang J J,Li J,et al.Activating transcription factor 4mediates hyperglycaemia-induced endothelial inflammation and retinal vascular leakage through activation of STAT3 in a mouse model of type 1 diabetes[J].Diabetologia,2012,55(9):2533-2545.
[14]李德山,姜兴昊,徐鹏飞,等.不同维持剂量聚乙二醇修饰的FGF-21在Ⅰ型糖尿病小鼠中降糖作用的研究[J].东北农业大学学报,2017(2):69-75.
[15]许会静,刘磊,董媛,等.FGF21对糖尿病Beagle犬降糖作用的研究[J].生物技术,2016(2):188-192.
[16]Xue C,Zhao J,Chen L,et al.Recent advances and state-of-theart strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum[J].Biotechnology Advances,2017,35(2):310-322.
[17]周海岩,刘龙,堵国成,等.诱导温度对大肠杆菌BR-42ΔpykA(p AP-B03)发酵生产L-苯丙氨酸的影响[J].食品与发酵工业,2014,40(4):7-12.
[18]Xu P,Ye X,Zhang Y,et al.Long-acting hypoglycemic effects of PEGylated FGF21 and insulin glargine in mice with type 1diabetes[J].Journal of Diabetes and its Complications,2015,29(1):5-12.
[19]何昆,张雅坤,叶贤龙,等.精氨酸修饰成纤维细胞生长因子21及修饰后蛋白体内稳定性和糖代谢调节作用的研究[J].生物化学与生物物理进展,2012,39(11):1089-1098.
[20]Thombre S,Gadgil M.Increase in efficiency of media utilization for recombinant protein production in Chinese hamster ovary culture through dilution[J].Biotechnology and Applied Biochemistry,2011,58(1):25-31.