长效促胰岛素降糖酵母的构建及其对糖尿病模型小鼠的治疗效果

doi:10.16288/j.yczz.14-206

遗传 ›› 2015, Vol. 37 ›› Issue (2): 183-191.doi: 10.16288/j.yczz.14-206

长效促胰岛素降糖酵母的构建及其对糖尿病模型小鼠的治疗效果

吴日¹,马超¹,李晓丹¹,段会坤¹,姬艳丽¹,王宇¹,姜苹哲¹,王海松¹,屠培培¹,李淼¹,尼钢钢¹,马百成²,李明刚¹

1. 南开大学生命科学学院,教育部生物活性材料重点实验室,天津 300071;
2. 天津市儿童医院,天津 300074

收稿日期:2014-06-25 出版日期:2015-02-20 发布日期:2015-01-19
通讯作者: 李明刚,教授,博士生导师,研究方向：生物技术与新药。E-mail: mgl@nankai.edu.cn
作者简介:吴日,教育部公派访问学者,金日成综合大学平壤医学院副教授。E-mail: tj20110901@163.com;马超,硕士研究生,专业方向：生物技术与新药。E-mail: mac1002@outlook.com;吴日和马超并列第一作者。
基金资助:
天津市科技支撑重点计划项目(编号：14ZCZDSY00013)资助

Construction of yeast strains expressing long-acting glucagon-like peptide-1 (GLP-1) and their therapeutic effects on type 2 diabetes mellitus mouse model

Ri Wu¹,Chao Ma¹,Xiaodan Li¹,Huikun Duan¹,Yanli Ji¹,Yu Wang¹,Pingzhe Jiang¹,Haisong Wang¹,Peipei Tu¹,Miao Li¹,Ganggang Ni¹,Baicheng Ma²,Minggang Li¹

1. Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, Tianjin 300071, China;
2. Tianjin Children’s Hospital, Tianjin 300074, China

Received:2014-06-25 Online:2015-02-20 Published:2015-01-19

摘要/Abstract

摘要： 益生菌生物药物是指通过口服表达药用多肽(蛋白)的重组益生菌活细胞达到治疗疾病的新型口服给药系统。为了构建一种能有效防治2型糖尿病的酵母生物药物,文章首先构建了酿酒酵母(S.cerevisiae)整合型表达载体pNK1-PGK,并且通过绿色荧光蛋白(GFP)证明其表达功能正常,利用该载体将10×GLP-1 (Glucagon-like peptide-1)基因转化到酿酒酵母INVSc1中,通过营养缺陷型和Western blotting成功筛选出表达10×GLP-1的长效促胰岛素降糖酵母(Long-acting GLP-1 hypoglycemic yeast, LHY)。该酵母生长迅速,外源基因10×GLP-1表达稳定,表达量达到1.56 mg/g细胞湿重。通过链脲佐菌素和高脂高糖饮食联合诱导的方法构建了2型糖尿病小鼠模型,用LHY对其进行口服灌胃治疗,证明LHY具有较好疗效,明显降低血糖水平。

关键词: 2型糖尿病, 胰高血糖素样肽-1(GLP-1), 长效促胰岛素降糖酵母(LHY), 重组酿酒酵母, 糖尿病小鼠模型

Abstract: Probiotics, i.e., bacteria expressing therapeutic peptides (protein), are used as a new type of orally administrated biologic drugs to treat diseases. To develop yeast strains which could effectively prevent and treat type 2 diabetes mellitus, we firstly constructed the yeast integrating plasmid pNK1-PGK which could successfully express green fluorescent protein (GFP) in Saccharomyces cerevisiae. The gene encoding ten tandem repeats of glucagon-like peptide-1(10×GLP-1) was cloned into the vector pNK1-PGK and the resulting plasmids were then transformed into the S. cerevisiae INVSc1. The long-acting GLP-1 hypoglycemic yeast (LHY) which grows rapidly and expresses 10×GLP-1 stably was selected by nutrition screening and Western blotting. The amount of 10×GLP-1 produced by LHY reached 1.56 mg per gram of wet cells. Moreover, the oral administration of LHY significantly reduced blood glucose level in type 2 diabetic mice induced by streptozotocin plus high fat and high sugar diet.

Key words: type 2 diabetes mellitus, glucagon-like peptide-1(GLP-1), long-acting GLP-1 hypoglycemic yeast (LHY), recombinant yeast, mouse model of type 2 diabetes mellitus

吴日,马超,李晓丹,段会坤,姬艳丽,王宇,姜苹哲,王海松,屠培培,李淼,尼钢钢,马百成,李明刚. 长效促胰岛素降糖酵母的构建及其对糖尿病模型小鼠的治疗效果[J]. 遗传, 2015, 37(2): 183-191.

Ri Wu,Chao Ma,Xiaodan Li,Huikun Duan,Yanli Ji,Yu Wang,Pingzhe Jiang,Haisong Wang,Peipei Tu,Miao Li,Ganggang Ni,Baicheng Ma,Minggang Li. Construction of yeast strains expressing long-acting glucagon-like peptide-1 (GLP-1) and their therapeutic effects on type 2 diabetes mellitus mouse model[J]. HEREDITAS(Beijing), 2015, 37(2): 183-191.

参考文献

[1] International Diabetes Federation. IDF diabetes atlas. 6th ed. Brussels, Belgium: International Diabetes Federation, 2013.
[2] Yang WY, Lu JM, Weng JP, Jia WP, Ji LN, Xiao JZ, Shan ZY, Liu J, Tian HM, Ji QH, Zhu DL, Ge JP, Lin XL, Chen L, Guo XH, Zhao ZG, Li Q, Zhou ZZ, Shan GL, He J, China National Diabetes and Metabolic Disorders Study Group. Prevalence of diabetes among men and women in China. N Engl J Med , 2010, 362(25): 1090-1101.
[3] Laurie LB, Daniel JD. Biology of incretins: GLP-1 and GIP. Gastroenterology , 2007, 132(6): 2131-2157.
[4] Hou JH, Yan RX, Ding DF, Yang LQ, Wang CY, Wu ZQ, Yu XJ, Li WD, Li MG. Oral administration of a fusion protein containing eight GLP- 1 analogues produced in Escherichia coli BL 21 (DE3) in streptozotocin- induced diabetic rats. Biotechno Lett , 2007, 29(10): 1439-1446.
[5] Hou JH, Yan RX, Yang LQ, Wu ZQ, Wang CY, Ding DF, Li N, Ma CX, Li MG. High-level expression of fusion protein containing 10 tandem repeated GLP-1 analogs in yeast Pichia Pastoris and its biological activity in a diabetic rat model. Biosci Biotechno Biochem , 2007, 71(6): 1462 -1469.
[6] Wu ZQ, Jia NB, Li N, Ma BC, Li MG. Construction of Saccharomyces cerevisiae strain stably expressing a fusion protein containing ten tandem recombinant human glucagon-like peptide-1 analogues. Chem Res Chinese U , 2009, 25(6): 882-886.
[7] Ma B, Tu P, Zhao X, Zhang Y, Wang Y, Ma C, Ji Y, Li X, Abbas SA, Li M. Expression and purification of optimized rolGLP-1, a novel GLP-1 analog, in Escherichia Coli BL21(DE3) and its good glucoregulatory effect on type 2 diabetic mice. Curr Pharm Biotechnol , 2013, 14(11): 985-994.
[8] 武志强, 贾耐兵, 李娜, 王瑞菊, 马百成, 王翠艳, 李明刚. 酵母整合型载体的构建及其功能分析. 生物学通报, 2008, 43(5): 47-50.
[9] Sambrook J, Russell DW, 黄培堂译. 分子克隆实验指南(第三版). 北京: 科学出版社, 2002.
[10] Amberg DC, 霍克克译. 酵母遗传学方法实验指南(第二版). 北京: 科学出版社, 2009.
[11] Yin X, Wei D, Yi L, Tao X, Ma Y. Expression and purification of exendin-4, a GLP-1 receptor agonist, in Escherichia coli . Protein Expr Purif , 2005, 41(2): 259-265.
[12] Lopes TS, de Wijs IJ, Steenhauer SI, Verbakel I, Planta RJ. Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae . Yeast , 1996, 12(5): 467-477.
[13] Okamoto T, Kanemoto N, Ohbuchi Y, Okano M, Fukui H, Sudo T. Characterization of STZ-induced type 2 diabetes in Zucker fatty rats. Exp Anim , 2008, 57(4): 335-345.
[14] Li Y, Tweedie D, Mattson MP, Holloway HW, Greig NH. Enhancing the GLP-1 receptor signaling pathway leads to proliferation and neuroprotection in human neuroblastoma cells. J Neurochem , 2010, 113(6): 1621-1631.

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长效促胰岛素降糖酵母的构建及其对糖尿病模型小鼠的治疗效果

Construction of yeast strains expressing long-acting glucagon-like peptide-1 (GLP-1) and their therapeutic effects on type 2 diabetes mellitus mouse model

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