长效融合蛋白GGH的构建、表达、纯化及其初步药效学和药代动力学
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摘要
胰高血糖素样多肽-1(GLP-1)是由肠道L细胞合成和分泌的由30个氨基酸组成的多肽。体内外研究显示GLP-1具有增强葡萄糖依赖性的胰岛素分泌、减少食物的摄取、减慢胃的排空、以及抑制胰高血糖素的分泌、刺激胰β细胞增殖、促进胰岛再生、以及抑制胰β细胞的凋亡等功能,且无胰岛素和磺脲类降糖药物的低血糖危险。GLP-1独特的降血糖作用机制,是现有抗糖尿病药物无可比拟的。但在体内其极易被二肽酰基肽酶Ⅳ降解,限制了GLP-1药物化的进展,本论文主要围绕GLP-1的长效性研究展开,主要结论如下:
利用PCR的方法克隆获得GLP-1及其突变体GLP-1_(A2G)的基因,并构建了大肠杆菌表达菌株BL21 (pGEX-4T-1-GLP-1)和BL21 (pGEX-4T-1-GLP-1_(A2G))。SDS-PAGE显示阳性克隆在分子量29.0 kDa处有明显的表达条带,与预测的分子量大小相近。凝血酶酶切后,进一步利用GST亲和层析与分子筛G75纯化获得了目标蛋白GLP-1和GLP-1_(A2G)。小鼠糖耐量实验证明,纯化获得的GLP-1和GLP-1_(A2G)在小鼠体内均有良好的控制血糖的生物活性,并且生物活性基本没有差异。
利用生物信息学软件InsightII、ICM pro、Amber计算得出GLP-1的突变体GLP-1_(A2G)与其突变体的串联体(GLP-1_(A2G))2的合理构象,并完成了其与GLP-1受体的分子对接,主要结合氨基酸残基为V30、L32、V36、Y69和P90,大部分是脂肪族和芳杂环类氨基酸,提示四个配体与nGLP-1R间的相互作用主要为疏水作用,并且计算得出GLP-1和突变体GLP-1_(A2G)的与受体结合能相差不大,而(GLP-1_(A2G))2与受体结合能大于GLP-1和突变体GLP-1_(A2G),说明(GLP-1_(A2G))2较GLP-1_(A2G)更适合与白蛋白融合表达。
利用重叠PCR技术,在体外成功拼接获得了(GLP-1_(A2G))2和HSA的融合基因(GGH),构建重组表达质粒pPIC9K-GGH,并将其通过电转化法转入毕赤酵母表达系统,经G418抗性筛选得到高表达菌株KM71(pPIC9K-GGH),在甲醇诱导下其表达量为162 mg/L, western-blot结果表明诱导表达的融合蛋白为GLP-1突变体和HSA的杂合分子。
对高表达菌株的发酵过程进行了优化,确定了毕赤酵母KM71(pPIC9K-GGH)的最佳生长条件:温度30℃、pH 6.0、装液量50-60 mL、甘油初始浓度4.0%、蛋白胨浓度2.0%。最佳表达条件:温度30℃、pH 6.0、装液量50-60 mL、诱导甲醇浓度2.0 %。在最优的条件下,毕赤酵母KM71(pPIC9K-GGH)菌体浓度最高达到21.0 g/L、最大比生长速率达到1.70 g/L·h、目标蛋白的最高产量达到245 mg/L、最大比产物生成速率达到3.38 mg/L,并且连续5个批次发酵水平稳定,批次之间的相对误差≤5%。
确定了融合蛋白GGH的分离纯化的主要步骤,即10000 r/min离心10 min,分子量10 kDa的biomax超滤膜浓缩20倍,大孔树脂DA101脱色,Q FF离子交换,Sephacryl S-200凝胶层析,整个纯化过程的回收率为50.1%。经SDS-PAGE和HPLC检测纯度大于95%,等电聚集测定等电点为4.6,符合下一步生物活性和药代动力学实验的要求。
融合蛋白GGH在体外对胰岛原代细胞生长有较好的刺激作用,在浓度45 nmoL/L时增殖率为35.4%,与单体的GLP-1相差不大。在糖耐量实验中,融合蛋白GGH可以较好的控制小鼠的血糖水平,并且在给药72 h后中、高剂量组仍然有控制血糖的生物活性,而单体的GLP-1在给药4 h后就检测不到生物活性。
药代动力学研究表明,融合蛋白GGH在腹腔皮下单次给药8 h左右,血药浓度达到最高,分布容积为53.4 mg/L·h,体内吸收半衰期为26.6 h,体内消除半衰期约为57.8 h。大鼠皮下注射该药后,主要脏器均于6 h达峰,小鼠的胃、肾、肺和肌肉等组织器官中单位重量放射性积聚较高,其中以胃为最高;在肠、胰腺、肝、生殖器和脂肪中单位重量的放射性活度次之,而在心和脑中的分布最少。300 h尿中平均累积排泄率为80.1%,说明肾脏是消除同位素的主要途径,间接反映出该药(包括代谢产物)主要通过肾排泄。300 h粪便中平均累积排泄率为16.0%,说明该药可部分通过粪便排泄。
Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. GLP-1 plays an important role in stimulating insulin secretion in a glucose induced manner, regulation of feeding, inhibiting glucagon secretion , increasingβcell proliferation, inhibitingβcell apoptosis and without low blood glucose dangerous with insulin and sulfonylureas hypoglycemic drugs. GLP-1 hypoglycemic unique mechanism is unparalleled to the existing anti-diabetes drugs. While, GLP-1 could easily degradated by DPPⅣin vitro, the progress to make GLP-1 as a drug was restricted. This study is mainly focused on long-term nature of GLP-1. The main conclusions are as follows:
PCR technology was employed to amplify the GLP-1and GLP-1_(A2G), and then the express strains BL21 (pGEX-4T-1-GLP-1) and BL21 (pGEX-4T-1-GLP-1_(A2G)) were constructed. The SDS-PAGE analysis showed the molecular weight of fusion protein GST- GLP-1 and GST- GLP-1_(A2G) was about 29.0KDa. The target protein of GLP-1 and GLP-1_(A2G) was obtained by GST affinity chromatography and Superdex G75 after digested with thrombin. The protein of GLP-1 and GLP-1_(A2G) show a good biological activity.
Bioinformatics softwares were used to calculated the rational conformations of GLP-1_(A2G) mutant and its concatenate, we also completed the two docking with the GLP-1 receptor molecule. The main binding amino acids were V30, L32, V36, Y69 and P90, most of which are aliphatic and heterocyclic amino acids, suggesting that the interaction between the four ligands and nGLP-1R is hydrophobic interaction. According calculating, there is no significated of acceptor binding energy between GLP-1 and GLP-1_(A2G), while the acceptor binding energy of (GLP-1_(A2G))_2 is larger than GLP-1 and GLP-1_(A2G), indicating that (GLP-1_(A2G))_2 is more fit for expression with albumin.
Overlap PCR technology was employed to amplify the fusion gene GGH, the construction of recombinant expression plasmid pPIC9K-GGH was electrotransformated to Pichia expression system. The high expression strain KM71(pPIC9K-GGH) was selected under G418 resistance, which production was 162 mg/L under the inducing with methanol. Using western-blot technology to identify the fusion protein is a GLP-1 and the HSA hybrid elements.
The fermentation process of high expression strain has been optimized, and the best growth condition of KM71(pPIC9K-GGH) has been determined: temperature 30℃, pH 6.0, liquid volume 50-60 mL in 500 mL flask,the initial concentration of glycerol 4.0%, the concentration of peptone 2.0%. The optimized expression condition consisted temperature 30℃, pH 6.0, liquid volume 50-60 mL in 500 mL flask, the methanol induced concentration 2.0 %. On the optimal condition, the highest cell concentration was 21.0 g/L, the largest specific growth rate reached 1.70 g/L·h, the highest production of target protein was up to 245 mg/L, and the best specific production formation rate was 3.38 mg/L·h. The result has been proved to be stable after five batches of fermentation, and the error of the five batches is no more than 5%.
The major steps of fusion protein separation and purification have been determined, first centrifuge at 10000 r/min 10 min, and then use biomax membrane (10 kDa mochular weight) concentration 20 times, to decolored with resin DA101 and use Q FF to ion exchange, gel chromatography was used by Sephacryl S-200. The recovery rate of purification in entire process is 50.1%. The purity detection through SDS-PAGE and HPLC was upper than 95%, this can be in line with pharmacokinetics request.
The fusion protein GGH can stimulate the growth of islet cell in vitro. When the concentration of GGH is 45 nmoL, the growth rate is 35.4%. The result was similar as GLP-1. In the glucose tolerance test, GGH fusion protein in mice can better control the blood sugar levels. After 72 h administration, biological activity of high-dose group still can be detected. However, there was no biological activity after 4 h administration GLP-1.that the, indirectly reflects 300 hours in was 16.0 percent, that part of the drug through fecal excretion.
Pharmacokinetics research showed that the maximum plasma concentration reached the highest after 8 h administration the fusion protein GGH by abdominal subcutaneous, the volume of distribution was 53.4 mg/L·h, the half-life of absorbtion in vivo was 26.6 h, the elimination half-life in vivo was about 57.8 h. Subcutaneous injection of the fusion protein GGH in mice, the main organs were all reached peak in 6 h, There were higher accumulations of radioactive by the unit weight in mice stomach, kidney, lung and muscle, especially it was highest in stomach, intestine, pancreas, liver, fat and genital were followed by. What’more, there had the least distribution in heart and brain.
The average cumulative fecal excretion rate in urine after 300 h was 80.1%. It is suggested that kidney was the main way to eliminate isotope, and also reflected that the drug (including metabolites) mainly egested through kidney. The average cumulative fecal excretion rate in manure after 300 h was 16.0%. It is suggested that this drug can egest by manure.
利用PCR的方法克隆获得GLP-1及其突变体GLP-1_(A2G)的基因,并构建了大肠杆菌表达菌株BL21 (pGEX-4T-1-GLP-1)和BL21 (pGEX-4T-1-GLP-1_(A2G))。SDS-PAGE显示阳性克隆在分子量29.0 kDa处有明显的表达条带,与预测的分子量大小相近。凝血酶酶切后,进一步利用GST亲和层析与分子筛G75纯化获得了目标蛋白GLP-1和GLP-1_(A2G)。小鼠糖耐量实验证明,纯化获得的GLP-1和GLP-1_(A2G)在小鼠体内均有良好的控制血糖的生物活性,并且生物活性基本没有差异。
利用生物信息学软件InsightII、ICM pro、Amber计算得出GLP-1的突变体GLP-1_(A2G)与其突变体的串联体(GLP-1_(A2G))2的合理构象,并完成了其与GLP-1受体的分子对接,主要结合氨基酸残基为V30、L32、V36、Y69和P90,大部分是脂肪族和芳杂环类氨基酸,提示四个配体与nGLP-1R间的相互作用主要为疏水作用,并且计算得出GLP-1和突变体GLP-1_(A2G)的与受体结合能相差不大,而(GLP-1_(A2G))2与受体结合能大于GLP-1和突变体GLP-1_(A2G),说明(GLP-1_(A2G))2较GLP-1_(A2G)更适合与白蛋白融合表达。
利用重叠PCR技术,在体外成功拼接获得了(GLP-1_(A2G))2和HSA的融合基因(GGH),构建重组表达质粒pPIC9K-GGH,并将其通过电转化法转入毕赤酵母表达系统,经G418抗性筛选得到高表达菌株KM71(pPIC9K-GGH),在甲醇诱导下其表达量为162 mg/L, western-blot结果表明诱导表达的融合蛋白为GLP-1突变体和HSA的杂合分子。
对高表达菌株的发酵过程进行了优化,确定了毕赤酵母KM71(pPIC9K-GGH)的最佳生长条件:温度30℃、pH 6.0、装液量50-60 mL、甘油初始浓度4.0%、蛋白胨浓度2.0%。最佳表达条件:温度30℃、pH 6.0、装液量50-60 mL、诱导甲醇浓度2.0 %。在最优的条件下,毕赤酵母KM71(pPIC9K-GGH)菌体浓度最高达到21.0 g/L、最大比生长速率达到1.70 g/L·h、目标蛋白的最高产量达到245 mg/L、最大比产物生成速率达到3.38 mg/L,并且连续5个批次发酵水平稳定,批次之间的相对误差≤5%。
确定了融合蛋白GGH的分离纯化的主要步骤,即10000 r/min离心10 min,分子量10 kDa的biomax超滤膜浓缩20倍,大孔树脂DA101脱色,Q FF离子交换,Sephacryl S-200凝胶层析,整个纯化过程的回收率为50.1%。经SDS-PAGE和HPLC检测纯度大于95%,等电聚集测定等电点为4.6,符合下一步生物活性和药代动力学实验的要求。
融合蛋白GGH在体外对胰岛原代细胞生长有较好的刺激作用,在浓度45 nmoL/L时增殖率为35.4%,与单体的GLP-1相差不大。在糖耐量实验中,融合蛋白GGH可以较好的控制小鼠的血糖水平,并且在给药72 h后中、高剂量组仍然有控制血糖的生物活性,而单体的GLP-1在给药4 h后就检测不到生物活性。
药代动力学研究表明,融合蛋白GGH在腹腔皮下单次给药8 h左右,血药浓度达到最高,分布容积为53.4 mg/L·h,体内吸收半衰期为26.6 h,体内消除半衰期约为57.8 h。大鼠皮下注射该药后,主要脏器均于6 h达峰,小鼠的胃、肾、肺和肌肉等组织器官中单位重量放射性积聚较高,其中以胃为最高;在肠、胰腺、肝、生殖器和脂肪中单位重量的放射性活度次之,而在心和脑中的分布最少。300 h尿中平均累积排泄率为80.1%,说明肾脏是消除同位素的主要途径,间接反映出该药(包括代谢产物)主要通过肾排泄。300 h粪便中平均累积排泄率为16.0%,说明该药可部分通过粪便排泄。
Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. GLP-1 plays an important role in stimulating insulin secretion in a glucose induced manner, regulation of feeding, inhibiting glucagon secretion , increasingβcell proliferation, inhibitingβcell apoptosis and without low blood glucose dangerous with insulin and sulfonylureas hypoglycemic drugs. GLP-1 hypoglycemic unique mechanism is unparalleled to the existing anti-diabetes drugs. While, GLP-1 could easily degradated by DPPⅣin vitro, the progress to make GLP-1 as a drug was restricted. This study is mainly focused on long-term nature of GLP-1. The main conclusions are as follows:
PCR technology was employed to amplify the GLP-1and GLP-1_(A2G), and then the express strains BL21 (pGEX-4T-1-GLP-1) and BL21 (pGEX-4T-1-GLP-1_(A2G)) were constructed. The SDS-PAGE analysis showed the molecular weight of fusion protein GST- GLP-1 and GST- GLP-1_(A2G) was about 29.0KDa. The target protein of GLP-1 and GLP-1_(A2G) was obtained by GST affinity chromatography and Superdex G75 after digested with thrombin. The protein of GLP-1 and GLP-1_(A2G) show a good biological activity.
Bioinformatics softwares were used to calculated the rational conformations of GLP-1_(A2G) mutant and its concatenate, we also completed the two docking with the GLP-1 receptor molecule. The main binding amino acids were V30, L32, V36, Y69 and P90, most of which are aliphatic and heterocyclic amino acids, suggesting that the interaction between the four ligands and nGLP-1R is hydrophobic interaction. According calculating, there is no significated of acceptor binding energy between GLP-1 and GLP-1_(A2G), while the acceptor binding energy of (GLP-1_(A2G))_2 is larger than GLP-1 and GLP-1_(A2G), indicating that (GLP-1_(A2G))_2 is more fit for expression with albumin.
Overlap PCR technology was employed to amplify the fusion gene GGH, the construction of recombinant expression plasmid pPIC9K-GGH was electrotransformated to Pichia expression system. The high expression strain KM71(pPIC9K-GGH) was selected under G418 resistance, which production was 162 mg/L under the inducing with methanol. Using western-blot technology to identify the fusion protein is a GLP-1 and the HSA hybrid elements.
The fermentation process of high expression strain has been optimized, and the best growth condition of KM71(pPIC9K-GGH) has been determined: temperature 30℃, pH 6.0, liquid volume 50-60 mL in 500 mL flask,the initial concentration of glycerol 4.0%, the concentration of peptone 2.0%. The optimized expression condition consisted temperature 30℃, pH 6.0, liquid volume 50-60 mL in 500 mL flask, the methanol induced concentration 2.0 %. On the optimal condition, the highest cell concentration was 21.0 g/L, the largest specific growth rate reached 1.70 g/L·h, the highest production of target protein was up to 245 mg/L, and the best specific production formation rate was 3.38 mg/L·h. The result has been proved to be stable after five batches of fermentation, and the error of the five batches is no more than 5%.
The major steps of fusion protein separation and purification have been determined, first centrifuge at 10000 r/min 10 min, and then use biomax membrane (10 kDa mochular weight) concentration 20 times, to decolored with resin DA101 and use Q FF to ion exchange, gel chromatography was used by Sephacryl S-200. The recovery rate of purification in entire process is 50.1%. The purity detection through SDS-PAGE and HPLC was upper than 95%, this can be in line with pharmacokinetics request.
The fusion protein GGH can stimulate the growth of islet cell in vitro. When the concentration of GGH is 45 nmoL, the growth rate is 35.4%. The result was similar as GLP-1. In the glucose tolerance test, GGH fusion protein in mice can better control the blood sugar levels. After 72 h administration, biological activity of high-dose group still can be detected. However, there was no biological activity after 4 h administration GLP-1.that the, indirectly reflects 300 hours in was 16.0 percent, that part of the drug through fecal excretion.
Pharmacokinetics research showed that the maximum plasma concentration reached the highest after 8 h administration the fusion protein GGH by abdominal subcutaneous, the volume of distribution was 53.4 mg/L·h, the half-life of absorbtion in vivo was 26.6 h, the elimination half-life in vivo was about 57.8 h. Subcutaneous injection of the fusion protein GGH in mice, the main organs were all reached peak in 6 h, There were higher accumulations of radioactive by the unit weight in mice stomach, kidney, lung and muscle, especially it was highest in stomach, intestine, pancreas, liver, fat and genital were followed by. What’more, there had the least distribution in heart and brain.
The average cumulative fecal excretion rate in urine after 300 h was 80.1%. It is suggested that kidney was the main way to eliminate isotope, and also reflected that the drug (including metabolites) mainly egested through kidney. The average cumulative fecal excretion rate in manure after 300 h was 16.0%. It is suggested that this drug can egest by manure.
引文
1.胡显文,马清钧.中国生物技术产业发展报告2005[M].北京:化学工业出社,2006.43-92.
2.戚楠,马清钧.长效重组蛋白药物的研究进展[J].中国生物工程杂志,2006,26(2):79-82
3.杨光英,孙正银.代谢综合征与2型糖尿病关系的研究进展[J].陕西医学杂志,2006,35(9):1167-1169
4.朱芸,刘金荣,张伟.降血糖药用植物资源概述[J].中西医结合学报,2004,2(1):67-68
5.朱禧星.现代糖尿病学[M].上海:上海医科大学出版社,2000.4-10
6.邵成雷,张玲.1型糖尿病治疗研究进展[J].广州医药,2004,35(1):6-9
7.周军,张志建,雷小宝.2型糖尿病治疗研究进展[J].邯郸医学高等专科学校学报,2005(18):356-358
8. Scheen A J. Drug treatment of non-insulin-dependent diabetes mellitus in the 1990s.Achievements and future developments[J].Drugs,1997,54(3):355-368
9.陈家伟.2型糖尿病降血糖药物应用的新进展[J].中华内分泌代谢杂志, 2005, 21(5):10-14
10. Langtly H D,Balfour J A, Langtry H D,et al.Glimepiride. A review of its use in the management of type 2 diabetes mellitue[J].Drugs,1998,55(4):563-571
11.任丽,赵衍斌,王媛.2型糖尿病的口服药物进展[J].2006,20(4):246-248
12. Hui H,Zhao X,Perfetti R.Structure and function studies of glucagon-like peptide-1 (GLP-1): the designing of a novel pharmacological agent for the treatment of diabetes[J].Diabetes Metab Res Rev,2005,21(2):313-331
13. Drucker D J.Minireview: the glucagon-like peptides[J].Endocrinology,2001,142(2): 521-527
14. Kreymann B,Williams G,Ghatei M A,et al.Glucagon-like peptide-1 7-36: a physiological incretin in man[J].Lancet,1987,2(8571):1300-1303
15. Vahl T P,Paty B W,Fuller B D,et al.Effects of GLP-1-(7-36)NH2, GLP-1-(7-37), and GLP-1- (9-36)NH2 on intravenous glucose tolerance and glucose-induced insulin secretion in healthy humans[J].Clin. Endocrinol. Metab,2003,88(4):1772-1779
16. Turton M D,O'Shea D,Gunn I,et al.A role for glucagon-like peptide-1 in the central regulation of feeding[J].Nature,1996,379(6560):69-72
17. Deacon C F,Wamberg S,Bie P,et al.Preservation of active incretin hormones by inhibition of dipeptidyl peptidase IV suppresses meal-induced incretin secretion in dogs[J].J Endocrinol.2002,172(2):355-62
18. Pospisilik J A,Stafford S G,Demuth H U, et al.Long-term treatment with the dipeptidyl peptidase IV inhibitor P32/ 98 causes sustained improvements in glucose tolerance, insulin sensitivity, hyperinsulinemia, and beta-cell glucoseresponsi-veness in VDF (fa/ fa) Zucker rats[J].Diabetes,2002,51(4):943- 950
19. Nauck M A,Niedereichholz U,Ettler R,et al.Glucagon-like peptide-1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans.Am J Physiol,1997,273(5):981-938
20. Orskov C,Rabenh?j L,Wettergren A, et al.Tissue and plasma concentrations of amidated and glycine-extended glucagon-like peptide I in humans[J].1994,43(4):535-9
21. Orskov C,Wettergren A,Holst JJ.Biological effects and metabolic rates of glucagonlike peptide-1 7-36 amide and glucagonlike peptide-1 7-37 in healthy subjects are indistinguishable[J].Diabetes.1993,42(5):658-61
22. Holst J J.Gut glucagon,enteroglucagon,gut GLI,glicentin -current status[J].Gastroentero- logy,1983,84(2):1602-1613
23. Holst J J,Bersani M, John A H,et al.Proglucagon processing in porcine and human pancreas[J].J Boil Chem,1994,269(29):18827-18833
24. Holst J J,Orskov C.Glucagon and other Proglucagon-derived peptides[J]. Raven Press, New York,1993.305-340
25. Orskov C,Rabenhoj L,Kofod H,et al.Production and secretion of amidated and glycine-extended glucagon-like peptide-1(GLP-1) in man[J].Diabetes,1994,43(2): 535-539
26. Sugiyama K,Mank H,Kato,et al.Sitmulation of truncated glucgon-like peptide-1 from the isolted perfused canine ileun by glucose absorption. Digestion, 1994,55:22-28.
27. Gunn I.Control of appetite the role of GLP-1(7-36) amide[J].Endocrnol,1997,155: 197-208.
28. Cancelas J, María L,Villanueva-Penacarrillo,et al.Potentiation and prolongation of the insulinotropic ction of glucogon-like peptide-1 by methyl pyruvate or dimethyl ester of L-glutamic cid in a type 2 diabetes animal model[J].Endocrine,2001,16(2):113-116.
29. Parker J C,Andews K M,Rescek,et al.Structure-fuction anaysis of a series of glucagons-like peptide-1 analogues[J].J Pept Res,1998,52:398-409
30. Neidigh J W,FesinmoLeyer R M,Prickett K S,et al.Exendin-4 and glucagons-like peptide-1:NMOLR structural comparsons in the solution and micelle-asssociated states[J].Biochemistry.2001,40(44):13188-13200
31.王毅飞,汪吉仪,王国华.胰高糖素样肽GL P21 (7236)NH2对β细胞内游离钙和胰岛素分泌的影响[J].中国糖尿病杂志,2001,9(1):57-58.
32. Farilla L,Bulotta A,Hirshberg B,et al.Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets[J]. Endocrinology.2003,144(12):5145-5148
33. Stoffer D A,Kiffer T J,Hussain M A,et a1.Insulinotropic glucagons-like peptide-1 agonists stimulate expression of homeodomainprotein IDX-1 and increase islet size in mouse pancreas[J].Diabetes,2000,49:741
34. Farilla L,Bulotta A,Hirshberg B,et a1.Glueagon-like peptide 1inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets[J].Endocrinology, 2003,144:149
35. Wang Q,Li XE, et a1.Glucagon-like peptide-1 regulates proliferation an d apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells.Diabetologia,2004.47:478
36. Adrian V,Pankaj S, Rita B, et a1.Efect of glucagom-like peptide-1(7-36)amide on initial splanchnic gIucose uptake and insulin action in humans with type 1 diabetes[J].Diabetes, 2001,50:565
37. Verdich C,Flint A,Gutzwiller J P. et a1. A meta-anulysis of the efect of glucagon-like peptide-1(7-36)amide on adlibitum energy intake in humans[J].J Clin Endocrinol Metab,2001,86:4382
38. Wilding J P,Smith D M,Ghatei M A,et a1.A role for glucagons-like peptide-1 in the central regulation of feeding[J].Natures,1996,379:69
39. Schick R R,Zimmermann J P, vorm Walde T,et al.Peptides that regulate food intake: glucagon-like peptide 1-(7-36) amide acts at lateral and medial hypothalamic sites to suppress feeding in rats[J].Am J Physiol Regul Integr Comp Physiol, 2003, 284(6): 427-35.
40. Thorens B.Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide-1[J]. Proc Natl Acad Sci USA, 1992, 89(18): 8641~8645
41. Bullock B P,Heller RS,Habener JF.Tissue distribution of messenger ribonucleic acid encoding the rat glucagon-like peptide-1 receptor[J].Endocrinology.1996,137(7): 2968-2978.
42. Tibaduiza E C,Chen C,Beinborn MA.small molecule ligand of the glucagon-like peptide-1 receptor targets its amino-terminal hormone binding domain[J].J Biol Chem, 2001,276(41):37787-37793
43.周彩红,胡慧,王明伟.胰高血糖素样肽1受体——治疗糖尿病新药的研究热点[J].生命科学,2004,16(2),90-95
44. Ikezawa Y,Yamatani K,Ohnuma H,et al.Glucagon-like peptide-1 inhibits glucagon-induced glycogenolysis in perivenous hepatocytes specifically[J].Regul Pept,2003,111(1-3):207~210.
45. Turton M D,O'Shea D,Gunn I,S. A.et al, A role for glucagon-like peptide-1 in the central regulation of feeding[J].Nature,1996,379:69-72
46. Zander M,Madsbad S,Madsen J L, Holst J J.Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2 diabetes: a parallel-group study.Lancet,2002,359(9):824-30
47. Pospisilik J A, Stafford S G , Demuth H U, et al. Long-term treatment with the dipeptidyl peptidase IV inhibitor P32/ 98 causes sustained improvements in glucose tolerance, insulin sensitivity, hyperinsulinemia, and beta-cell glucoseresponsi-veness in VDF (fa/ fa) Zucker rats[J].Diabetes,2002,51:943- 950
48. Deacon C F,.Johnsen A H, Holst J J,Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo[J],J. Clin. Endocrinol.Metab,1995,80:952-957
49.张志珍,刘智慧.人胰高血糖素样肽-1突变体基因的克隆及表达[J].生物技术,2004,14(3):4-7
50.张志珍,毛积芳,杨生生.重组人胰高血糖素样肽-1对实验性糖尿病大鼠血糖的影响.中国生化药物杂志,2004,25(5):287-289
51. Lee S H,Lee S,Youn Y S,et al.Synthesis, characterization, and pharmacok inetic studies of PEGylated glucagons-like pep tide-1[J].Bioconjug Chem,2005,16(2):377
52. Claus B,Juhl M H,Jeppe S,et al.Bedtime Administration of NN2211, a Long-Acting GLP-1 Derivative, Substantially Reduces Fasting and Postprandial Glycemia in Type 2 Diabetes[J].Diabetes,2002,51:424-429,
53.卢曦,张城.治疗2型糖尿病的新颖化合物胰高血糖素样肽-1衍生物: NN2211在临床上的发展[J].国外医学·药学分册,2002,29(4):228-231
54. Kim J G,Baggio L L,Bridon D P,et al.Development and characterization of a glucagons-like peptide-1 album in conjugate[J].Diabetes,2003,52:751-759
55.葛晓宇,刘景晶.Exendin-4及其类似物的研究近况[J].2007,31(9):403-407
56. Gang X,Doris A S,Joel F H,et al.Exendin-4 stimulates bothβ-cell replication and neogenesis, resulting in increasedβ-cell mass and improved glucose toerence in diabetic rats[J].Diabetes,1999,48:2270-2276
57. Orville G. Kolterman,John B,et al.Synthetic Exendin-4 (Exenatide) Significantly Reduces Postprandial and Fasting Plasma Glucose in Subjects with Type 2 Diabetes[J].The Journal of Clinical Endocrinology & Metabolism,2003,88(7): 3082-3089
58. Egan J M,Clocquet A,Elahi D.The insulino trop ic effect of acute exendin24 adm inistered to humans comparison of nondiabetic state to type 2 diabetes[J].J Cl in Endocrino lM etab,2002,87(3):1282
59. Choi S,Baudys M,Kim S,et al.Control of blood glucose by novel GLP-1 delivery using biodegradable triblock copolymer of PL GA 2PEG2PL GA in type 2 diabetic rats[J]. Pharmaceutical Research,2004,21(5):827
60.李慧,陈家琪.胰高血糖素样肽1类似物的研究进展[J].天津药学,2005,17(3):41-43
61. Ahrén B,Winzell M S,Wierup N,et al.DPP-4 inhibition improves glucose tolerance and increases insulin and GLP-1 responses to gastric glucose in association with normalized islet topography in mice with beta-cell-specific overexpression of human islet amyloid polypeptide[J].Regul. Pept.2007,143(1):97-103
62.梁文昌.二羧基肽酶DPP-4抑制剂治疗糖尿病的研究进展[J].国外医学·内分泌学分册, 2004,24(1):58-61
63. Bo A,Erik S,HilleviL,et al.Inhibition of dipeptidyl peptidase 4 rovesmetabolic control over a 42 week study period in type 2 diabetes[J].Diabetes Care,2002,25:869
65. Lucas L H,Price K E,Larive C K.Epitope mapping and competitive binding of HSA drug site II ligands by NMOLR diffusion measurements[J].J Am Chem Soc,2004,126 (43):14258-14266
66. Petes T P.All About Albumin[M].San Diego:Academic Press,1996.
67. Kobayashi K,Nakamura N,Sumi A,et al.The development ofrecombinant human serum albumin[J].Ther Apher,1998,2(4):257-262
68. P Yeh,D Landais,M Lema?tre,et al.Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate[J].Proc Natl Acad Sci.1992,89(5):1904–1908
70. Osborn B L,Olsen H S,Nardelli B,et al.Pharmacokinetic and pharmacodynamic studies of a human serum albumin2interferon2alpha fusion p rotein in cynomolgus monkeys[J].J Pharmacol Exp Ther,2002,303(2):540-548
71. Human Genome Sciences Reports Positive Results of Phase 1/2 Clinical Trial of Albuferon in Chronic HepatitisC.HGSI Press,2004,11.2
71.王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志.2003,23(10):
72.雷楗勇,张莲芬,金坚,等.人β干扰素-血清白蛋白融合蛋白在毕赤酵母中的分泌表达[J].中国生物工程杂志,2006,26(7):13-18
73.徐钰,孙钧铭,张莲芬,等.人GCSF-白蛋白融合蛋白的构建及在毕赤酵母中的分泌表达[J].药物生物技术,2008,15(2):109-114
74.李洁,张莲芬,孙钧铭,等.人C2型利钠肽2血清白蛋白融合蛋白质的构建及在毕赤酵母中的分泌表达[J].中国生化药物杂志,2008,28(5):289-293
75. Choi J H,Lee S Y.Secretory and extracellular production of recombinant proteins using Escherichia coli[J].Appl Microbiol Biotechnol,2004,64(5):625-635
76.肖洁,郭刚,邹全明.提高大肠杆菌分泌表达重组蛋白的研究进展[J].微生物学杂志,2007,27(2):73-77
77.郑晶,陆惠玲,刘加军.大肠杆菌表达系统的研究进展药[J].物生物技术,2005,12(6):416-420
78. J.萨姆布鲁克.分子克隆实验指南[M]:上册.第三版.北京:科学出版社,2002:40-60
79.胥俊峰,单建华,赵宁伟,等.大肠杆菌γ-ggt原核表达载体pGEX-4T-1/γ2ggt的构建及其蛋白表达[J].2007,35(30):9467-9469
80.周宇荀,魏东芝,王二力.融合蛋白表达载体pGEX及其应用[J].生命科学,1998,10(3):122-124
81. Koti S,Robert G,Brankamp,et al.Strategies for optimal protein expression and secretion in the methylotrophic yeast Pichia.gene,1997,190(1):55-62
82. Mar C Andrew J D.A rational approach to improving productivity in recombinant Pichia pastoris fermentation [J].Bioteeh BioEng,2001,72(1):1-11
83. Hasslacher M,Schall M,Hayn M.High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J].Protein Expression and Purification,1997,119(1):61-71
84.李健仔.巴斯德毕赤酵母外源基因表达系统[J].生物学通报,2005,40(3):21-23
85.王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志,23(10),23-27
86.奥斯伯,布伦特,金斯,等.顿新编分子生物学指南[M].2001,北京:509-518
87. Invitrogen. Pichia Expression Kit:A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris.
88. Sreekrishna K,Brankamp R G, Kropp K E,et al.Strategies for optimal synthesis of heterologous proteins in the methylotrophic yeast Pichia pastoris [J].Gene,1997,190:55-62
89.龙晶,杜立新.巴斯德毕赤酵母表达外源蛋白的研究进展[J].微生物学杂志2007,27(6):72-76
90.张宏斌,窦文芳,许泓瑜,等.提高重组毕赤酵母表达hIFNβ-HSA的碳源控制策略过[J].过程工程学报,2007,6(7):1182-1186
91. Zhuge J,Fang H Y,Wang Z X.Glycerol Production by a Novel Osmotolerant YeastCandida glycerinogenes [J]. Applied Microbiology and Biotechnology,2001,55:686-692
92. Jayanta S,Bradley A P,Mehmet I,et al.Causes of Proteolytic Degradation of Secreted Recombinant Proteins Produced in Methylotrophic Yeast Pichia pastoris: Case Study with Recombinant Ovine Interferon-T [J].Biotechnol.Bioeng,2005,89(1):103-112
93. Minning S,Schmidt-Dannert C, Schmid R D. Functional Expression of Rhizopus oryzae Lipase in Pichia pastoris: High-level Production and Some Properties [J].J Biotechnol,1998,66:147-156
94. Mehmet I,Vijay C,Kent M,et al.Optimization of temperature glycerol pH conditions for a fed-batch fermentation process for recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (AcAP-5) production by Pichia pastoris[J].Enzyme and Microbial Technology,1999,24(7):438-445
95. Carmen J,Ian M,Urs S.Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: A quantitative study based on concentration gradients in transient continuous cultures Journal of Biotechnology[J].2007,128(4): 824-837
96. Rodriguez-Jimenez E,Sanchez K,Roca H,et al.Different Methanol Feeding Strategies to Recombinant Pichia pastoris Cultures Producing High Level of Dextranase[J]. Biotechnol Techniques,1997,11:461-466
97.钟宁,王佩迪.膜技术在中药提取分离、制备中的应用[J].安徽医药,2007,11(01):.
98.李淑莉,刘振理,宋志前.超滤法在中药制剂纯化工艺中的应用研究进展[J].2003,26(3):898-900
99.陶永辉.重组人血管抑素的制备及其作用特异性的研究[C].江南大学博士学位论文,2005
100. Kobayashi K, Kuwae S, Ohya T, ey al. High-Level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation[J].J.Biosci.Bioeng.2000,89:51-61
101.王毅飞王洁仪胰高糖素样肽GLP-1(7-36)NH2对体外培养的新生胰岛细胞作用的研究[J].1999,19(5):411-413
102. Laurie LB,Qingling H,1 Theodore J,et al.Recombinant Human Glucagon-Like Peptide(GLP)-1–Albumin Protein (Albugon) MimicsPeptidergic Activation of GLP-1 Receptor–DependentPathways Coupled With Satiety, GastrointestinalMotility, and Glucose Homeostasis[J].Diabetes,2004,53:2492-2500
103.龙祯,蒋丽华,华欲飞.大孔树脂对大豆糖蜜超滤液的脱色效果研究[J].中国油脂,2008(2):53-56
104.江力宣,闫海英,阳盛洪,等.药代动力学在新药研发中的应用[J].药学实践杂志,2006,24(4):261-263
105.樊蓉,张纯,高申.蛋白多肽类药物研究概况[J].药学实践杂志,2006,24(3):135-138
106.王军志.生物技术药物研究开发和质量控制[M].北京:科学技术出版社,2002
107. Beom S S,Chul H K,Min-Nyung L,et al.Pharmacokinetics of 125I-GST-TatdMt, a recombinant fusion proteinpossessing potent anti-obesity activity, after intravenous,nasal, oral, and subcutaneous administration[J].Regulatory Peptides,2007(140):74-80
108.廖建民,张奉国,常高翔,等.125 I示踪法研究rhPTH(1-34)在大鼠体内的药代动力学[J].同位素,2006,19(4):223-227
109.邢文会,杨萍,汪海霞,等.猕猴体内白蛋白融合干扰素α-2b( HSA-IFNα-2b)的药代动力学研究中国药理学通报[J].2007,23(9):1156-1160
110 Huang Y S,Chen Z H,Yang Z Y,et al.Preparation and characterization of a potent, long-lasting recombinant human serum albumin-interferon-α2b fusion protein expressed in Pichia pastoris[J].Eur.J.Pharm.Biopharm,2007,67:301-308
2.戚楠,马清钧.长效重组蛋白药物的研究进展[J].中国生物工程杂志,2006,26(2):79-82
3.杨光英,孙正银.代谢综合征与2型糖尿病关系的研究进展[J].陕西医学杂志,2006,35(9):1167-1169
4.朱芸,刘金荣,张伟.降血糖药用植物资源概述[J].中西医结合学报,2004,2(1):67-68
5.朱禧星.现代糖尿病学[M].上海:上海医科大学出版社,2000.4-10
6.邵成雷,张玲.1型糖尿病治疗研究进展[J].广州医药,2004,35(1):6-9
7.周军,张志建,雷小宝.2型糖尿病治疗研究进展[J].邯郸医学高等专科学校学报,2005(18):356-358
8. Scheen A J. Drug treatment of non-insulin-dependent diabetes mellitus in the 1990s.Achievements and future developments[J].Drugs,1997,54(3):355-368
9.陈家伟.2型糖尿病降血糖药物应用的新进展[J].中华内分泌代谢杂志, 2005, 21(5):10-14
10. Langtly H D,Balfour J A, Langtry H D,et al.Glimepiride. A review of its use in the management of type 2 diabetes mellitue[J].Drugs,1998,55(4):563-571
11.任丽,赵衍斌,王媛.2型糖尿病的口服药物进展[J].2006,20(4):246-248
12. Hui H,Zhao X,Perfetti R.Structure and function studies of glucagon-like peptide-1 (GLP-1): the designing of a novel pharmacological agent for the treatment of diabetes[J].Diabetes Metab Res Rev,2005,21(2):313-331
13. Drucker D J.Minireview: the glucagon-like peptides[J].Endocrinology,2001,142(2): 521-527
14. Kreymann B,Williams G,Ghatei M A,et al.Glucagon-like peptide-1 7-36: a physiological incretin in man[J].Lancet,1987,2(8571):1300-1303
15. Vahl T P,Paty B W,Fuller B D,et al.Effects of GLP-1-(7-36)NH2, GLP-1-(7-37), and GLP-1- (9-36)NH2 on intravenous glucose tolerance and glucose-induced insulin secretion in healthy humans[J].Clin. Endocrinol. Metab,2003,88(4):1772-1779
16. Turton M D,O'Shea D,Gunn I,et al.A role for glucagon-like peptide-1 in the central regulation of feeding[J].Nature,1996,379(6560):69-72
17. Deacon C F,Wamberg S,Bie P,et al.Preservation of active incretin hormones by inhibition of dipeptidyl peptidase IV suppresses meal-induced incretin secretion in dogs[J].J Endocrinol.2002,172(2):355-62
18. Pospisilik J A,Stafford S G,Demuth H U, et al.Long-term treatment with the dipeptidyl peptidase IV inhibitor P32/ 98 causes sustained improvements in glucose tolerance, insulin sensitivity, hyperinsulinemia, and beta-cell glucoseresponsi-veness in VDF (fa/ fa) Zucker rats[J].Diabetes,2002,51(4):943- 950
19. Nauck M A,Niedereichholz U,Ettler R,et al.Glucagon-like peptide-1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans.Am J Physiol,1997,273(5):981-938
20. Orskov C,Rabenh?j L,Wettergren A, et al.Tissue and plasma concentrations of amidated and glycine-extended glucagon-like peptide I in humans[J].1994,43(4):535-9
21. Orskov C,Wettergren A,Holst JJ.Biological effects and metabolic rates of glucagonlike peptide-1 7-36 amide and glucagonlike peptide-1 7-37 in healthy subjects are indistinguishable[J].Diabetes.1993,42(5):658-61
22. Holst J J.Gut glucagon,enteroglucagon,gut GLI,glicentin -current status[J].Gastroentero- logy,1983,84(2):1602-1613
23. Holst J J,Bersani M, John A H,et al.Proglucagon processing in porcine and human pancreas[J].J Boil Chem,1994,269(29):18827-18833
24. Holst J J,Orskov C.Glucagon and other Proglucagon-derived peptides[J]. Raven Press, New York,1993.305-340
25. Orskov C,Rabenhoj L,Kofod H,et al.Production and secretion of amidated and glycine-extended glucagon-like peptide-1(GLP-1) in man[J].Diabetes,1994,43(2): 535-539
26. Sugiyama K,Mank H,Kato,et al.Sitmulation of truncated glucgon-like peptide-1 from the isolted perfused canine ileun by glucose absorption. Digestion, 1994,55:22-28.
27. Gunn I.Control of appetite the role of GLP-1(7-36) amide[J].Endocrnol,1997,155: 197-208.
28. Cancelas J, María L,Villanueva-Penacarrillo,et al.Potentiation and prolongation of the insulinotropic ction of glucogon-like peptide-1 by methyl pyruvate or dimethyl ester of L-glutamic cid in a type 2 diabetes animal model[J].Endocrine,2001,16(2):113-116.
29. Parker J C,Andews K M,Rescek,et al.Structure-fuction anaysis of a series of glucagons-like peptide-1 analogues[J].J Pept Res,1998,52:398-409
30. Neidigh J W,FesinmoLeyer R M,Prickett K S,et al.Exendin-4 and glucagons-like peptide-1:NMOLR structural comparsons in the solution and micelle-asssociated states[J].Biochemistry.2001,40(44):13188-13200
31.王毅飞,汪吉仪,王国华.胰高糖素样肽GL P21 (7236)NH2对β细胞内游离钙和胰岛素分泌的影响[J].中国糖尿病杂志,2001,9(1):57-58.
32. Farilla L,Bulotta A,Hirshberg B,et al.Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets[J]. Endocrinology.2003,144(12):5145-5148
33. Stoffer D A,Kiffer T J,Hussain M A,et a1.Insulinotropic glucagons-like peptide-1 agonists stimulate expression of homeodomainprotein IDX-1 and increase islet size in mouse pancreas[J].Diabetes,2000,49:741
34. Farilla L,Bulotta A,Hirshberg B,et a1.Glueagon-like peptide 1inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets[J].Endocrinology, 2003,144:149
35. Wang Q,Li XE, et a1.Glucagon-like peptide-1 regulates proliferation an d apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells.Diabetologia,2004.47:478
36. Adrian V,Pankaj S, Rita B, et a1.Efect of glucagom-like peptide-1(7-36)amide on initial splanchnic gIucose uptake and insulin action in humans with type 1 diabetes[J].Diabetes, 2001,50:565
37. Verdich C,Flint A,Gutzwiller J P. et a1. A meta-anulysis of the efect of glucagon-like peptide-1(7-36)amide on adlibitum energy intake in humans[J].J Clin Endocrinol Metab,2001,86:4382
38. Wilding J P,Smith D M,Ghatei M A,et a1.A role for glucagons-like peptide-1 in the central regulation of feeding[J].Natures,1996,379:69
39. Schick R R,Zimmermann J P, vorm Walde T,et al.Peptides that regulate food intake: glucagon-like peptide 1-(7-36) amide acts at lateral and medial hypothalamic sites to suppress feeding in rats[J].Am J Physiol Regul Integr Comp Physiol, 2003, 284(6): 427-35.
40. Thorens B.Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide-1[J]. Proc Natl Acad Sci USA, 1992, 89(18): 8641~8645
41. Bullock B P,Heller RS,Habener JF.Tissue distribution of messenger ribonucleic acid encoding the rat glucagon-like peptide-1 receptor[J].Endocrinology.1996,137(7): 2968-2978.
42. Tibaduiza E C,Chen C,Beinborn MA.small molecule ligand of the glucagon-like peptide-1 receptor targets its amino-terminal hormone binding domain[J].J Biol Chem, 2001,276(41):37787-37793
43.周彩红,胡慧,王明伟.胰高血糖素样肽1受体——治疗糖尿病新药的研究热点[J].生命科学,2004,16(2),90-95
44. Ikezawa Y,Yamatani K,Ohnuma H,et al.Glucagon-like peptide-1 inhibits glucagon-induced glycogenolysis in perivenous hepatocytes specifically[J].Regul Pept,2003,111(1-3):207~210.
45. Turton M D,O'Shea D,Gunn I,S. A.et al, A role for glucagon-like peptide-1 in the central regulation of feeding[J].Nature,1996,379:69-72
46. Zander M,Madsbad S,Madsen J L, Holst J J.Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2 diabetes: a parallel-group study.Lancet,2002,359(9):824-30
47. Pospisilik J A, Stafford S G , Demuth H U, et al. Long-term treatment with the dipeptidyl peptidase IV inhibitor P32/ 98 causes sustained improvements in glucose tolerance, insulin sensitivity, hyperinsulinemia, and beta-cell glucoseresponsi-veness in VDF (fa/ fa) Zucker rats[J].Diabetes,2002,51:943- 950
48. Deacon C F,.Johnsen A H, Holst J J,Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo[J],J. Clin. Endocrinol.Metab,1995,80:952-957
49.张志珍,刘智慧.人胰高血糖素样肽-1突变体基因的克隆及表达[J].生物技术,2004,14(3):4-7
50.张志珍,毛积芳,杨生生.重组人胰高血糖素样肽-1对实验性糖尿病大鼠血糖的影响.中国生化药物杂志,2004,25(5):287-289
51. Lee S H,Lee S,Youn Y S,et al.Synthesis, characterization, and pharmacok inetic studies of PEGylated glucagons-like pep tide-1[J].Bioconjug Chem,2005,16(2):377
52. Claus B,Juhl M H,Jeppe S,et al.Bedtime Administration of NN2211, a Long-Acting GLP-1 Derivative, Substantially Reduces Fasting and Postprandial Glycemia in Type 2 Diabetes[J].Diabetes,2002,51:424-429,
53.卢曦,张城.治疗2型糖尿病的新颖化合物胰高血糖素样肽-1衍生物: NN2211在临床上的发展[J].国外医学·药学分册,2002,29(4):228-231
54. Kim J G,Baggio L L,Bridon D P,et al.Development and characterization of a glucagons-like peptide-1 album in conjugate[J].Diabetes,2003,52:751-759
55.葛晓宇,刘景晶.Exendin-4及其类似物的研究近况[J].2007,31(9):403-407
56. Gang X,Doris A S,Joel F H,et al.Exendin-4 stimulates bothβ-cell replication and neogenesis, resulting in increasedβ-cell mass and improved glucose toerence in diabetic rats[J].Diabetes,1999,48:2270-2276
57. Orville G. Kolterman,John B,et al.Synthetic Exendin-4 (Exenatide) Significantly Reduces Postprandial and Fasting Plasma Glucose in Subjects with Type 2 Diabetes[J].The Journal of Clinical Endocrinology & Metabolism,2003,88(7): 3082-3089
58. Egan J M,Clocquet A,Elahi D.The insulino trop ic effect of acute exendin24 adm inistered to humans comparison of nondiabetic state to type 2 diabetes[J].J Cl in Endocrino lM etab,2002,87(3):1282
59. Choi S,Baudys M,Kim S,et al.Control of blood glucose by novel GLP-1 delivery using biodegradable triblock copolymer of PL GA 2PEG2PL GA in type 2 diabetic rats[J]. Pharmaceutical Research,2004,21(5):827
60.李慧,陈家琪.胰高血糖素样肽1类似物的研究进展[J].天津药学,2005,17(3):41-43
61. Ahrén B,Winzell M S,Wierup N,et al.DPP-4 inhibition improves glucose tolerance and increases insulin and GLP-1 responses to gastric glucose in association with normalized islet topography in mice with beta-cell-specific overexpression of human islet amyloid polypeptide[J].Regul. Pept.2007,143(1):97-103
62.梁文昌.二羧基肽酶DPP-4抑制剂治疗糖尿病的研究进展[J].国外医学·内分泌学分册, 2004,24(1):58-61
63. Bo A,Erik S,HilleviL,et al.Inhibition of dipeptidyl peptidase 4 rovesmetabolic control over a 42 week study period in type 2 diabetes[J].Diabetes Care,2002,25:869
65. Lucas L H,Price K E,Larive C K.Epitope mapping and competitive binding of HSA drug site II ligands by NMOLR diffusion measurements[J].J Am Chem Soc,2004,126 (43):14258-14266
66. Petes T P.All About Albumin[M].San Diego:Academic Press,1996.
67. Kobayashi K,Nakamura N,Sumi A,et al.The development ofrecombinant human serum albumin[J].Ther Apher,1998,2(4):257-262
68. P Yeh,D Landais,M Lema?tre,et al.Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate[J].Proc Natl Acad Sci.1992,89(5):1904–1908
70. Osborn B L,Olsen H S,Nardelli B,et al.Pharmacokinetic and pharmacodynamic studies of a human serum albumin2interferon2alpha fusion p rotein in cynomolgus monkeys[J].J Pharmacol Exp Ther,2002,303(2):540-548
71. Human Genome Sciences Reports Positive Results of Phase 1/2 Clinical Trial of Albuferon in Chronic HepatitisC.HGSI Press,2004,11.2
71.王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志.2003,23(10):
72.雷楗勇,张莲芬,金坚,等.人β干扰素-血清白蛋白融合蛋白在毕赤酵母中的分泌表达[J].中国生物工程杂志,2006,26(7):13-18
73.徐钰,孙钧铭,张莲芬,等.人GCSF-白蛋白融合蛋白的构建及在毕赤酵母中的分泌表达[J].药物生物技术,2008,15(2):109-114
74.李洁,张莲芬,孙钧铭,等.人C2型利钠肽2血清白蛋白融合蛋白质的构建及在毕赤酵母中的分泌表达[J].中国生化药物杂志,2008,28(5):289-293
75. Choi J H,Lee S Y.Secretory and extracellular production of recombinant proteins using Escherichia coli[J].Appl Microbiol Biotechnol,2004,64(5):625-635
76.肖洁,郭刚,邹全明.提高大肠杆菌分泌表达重组蛋白的研究进展[J].微生物学杂志,2007,27(2):73-77
77.郑晶,陆惠玲,刘加军.大肠杆菌表达系统的研究进展药[J].物生物技术,2005,12(6):416-420
78. J.萨姆布鲁克.分子克隆实验指南[M]:上册.第三版.北京:科学出版社,2002:40-60
79.胥俊峰,单建华,赵宁伟,等.大肠杆菌γ-ggt原核表达载体pGEX-4T-1/γ2ggt的构建及其蛋白表达[J].2007,35(30):9467-9469
80.周宇荀,魏东芝,王二力.融合蛋白表达载体pGEX及其应用[J].生命科学,1998,10(3):122-124
81. Koti S,Robert G,Brankamp,et al.Strategies for optimal protein expression and secretion in the methylotrophic yeast Pichia.gene,1997,190(1):55-62
82. Mar C Andrew J D.A rational approach to improving productivity in recombinant Pichia pastoris fermentation [J].Bioteeh BioEng,2001,72(1):1-11
83. Hasslacher M,Schall M,Hayn M.High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J].Protein Expression and Purification,1997,119(1):61-71
84.李健仔.巴斯德毕赤酵母外源基因表达系统[J].生物学通报,2005,40(3):21-23
85.王秀贞,吴军,孟宪军.长效多肽药物研究进展[J].中国生物工程杂志,23(10),23-27
86.奥斯伯,布伦特,金斯,等.顿新编分子生物学指南[M].2001,北京:509-518
87. Invitrogen. Pichia Expression Kit:A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris.
88. Sreekrishna K,Brankamp R G, Kropp K E,et al.Strategies for optimal synthesis of heterologous proteins in the methylotrophic yeast Pichia pastoris [J].Gene,1997,190:55-62
89.龙晶,杜立新.巴斯德毕赤酵母表达外源蛋白的研究进展[J].微生物学杂志2007,27(6):72-76
90.张宏斌,窦文芳,许泓瑜,等.提高重组毕赤酵母表达hIFNβ-HSA的碳源控制策略过[J].过程工程学报,2007,6(7):1182-1186
91. Zhuge J,Fang H Y,Wang Z X.Glycerol Production by a Novel Osmotolerant YeastCandida glycerinogenes [J]. Applied Microbiology and Biotechnology,2001,55:686-692
92. Jayanta S,Bradley A P,Mehmet I,et al.Causes of Proteolytic Degradation of Secreted Recombinant Proteins Produced in Methylotrophic Yeast Pichia pastoris: Case Study with Recombinant Ovine Interferon-T [J].Biotechnol.Bioeng,2005,89(1):103-112
93. Minning S,Schmidt-Dannert C, Schmid R D. Functional Expression of Rhizopus oryzae Lipase in Pichia pastoris: High-level Production and Some Properties [J].J Biotechnol,1998,66:147-156
94. Mehmet I,Vijay C,Kent M,et al.Optimization of temperature glycerol pH conditions for a fed-batch fermentation process for recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (AcAP-5) production by Pichia pastoris[J].Enzyme and Microbial Technology,1999,24(7):438-445
95. Carmen J,Ian M,Urs S.Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: A quantitative study based on concentration gradients in transient continuous cultures Journal of Biotechnology[J].2007,128(4): 824-837
96. Rodriguez-Jimenez E,Sanchez K,Roca H,et al.Different Methanol Feeding Strategies to Recombinant Pichia pastoris Cultures Producing High Level of Dextranase[J]. Biotechnol Techniques,1997,11:461-466
97.钟宁,王佩迪.膜技术在中药提取分离、制备中的应用[J].安徽医药,2007,11(01):.
98.李淑莉,刘振理,宋志前.超滤法在中药制剂纯化工艺中的应用研究进展[J].2003,26(3):898-900
99.陶永辉.重组人血管抑素的制备及其作用特异性的研究[C].江南大学博士学位论文,2005
100. Kobayashi K, Kuwae S, Ohya T, ey al. High-Level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation[J].J.Biosci.Bioeng.2000,89:51-61
101.王毅飞王洁仪胰高糖素样肽GLP-1(7-36)NH2对体外培养的新生胰岛细胞作用的研究[J].1999,19(5):411-413
102. Laurie LB,Qingling H,1 Theodore J,et al.Recombinant Human Glucagon-Like Peptide(GLP)-1–Albumin Protein (Albugon) MimicsPeptidergic Activation of GLP-1 Receptor–DependentPathways Coupled With Satiety, GastrointestinalMotility, and Glucose Homeostasis[J].Diabetes,2004,53:2492-2500
103.龙祯,蒋丽华,华欲飞.大孔树脂对大豆糖蜜超滤液的脱色效果研究[J].中国油脂,2008(2):53-56
104.江力宣,闫海英,阳盛洪,等.药代动力学在新药研发中的应用[J].药学实践杂志,2006,24(4):261-263
105.樊蓉,张纯,高申.蛋白多肽类药物研究概况[J].药学实践杂志,2006,24(3):135-138
106.王军志.生物技术药物研究开发和质量控制[M].北京:科学技术出版社,2002
107. Beom S S,Chul H K,Min-Nyung L,et al.Pharmacokinetics of 125I-GST-TatdMt, a recombinant fusion proteinpossessing potent anti-obesity activity, after intravenous,nasal, oral, and subcutaneous administration[J].Regulatory Peptides,2007(140):74-80
108.廖建民,张奉国,常高翔,等.125 I示踪法研究rhPTH(1-34)在大鼠体内的药代动力学[J].同位素,2006,19(4):223-227
109.邢文会,杨萍,汪海霞,等.猕猴体内白蛋白融合干扰素α-2b( HSA-IFNα-2b)的药代动力学研究中国药理学通报[J].2007,23(9):1156-1160
110 Huang Y S,Chen Z H,Yang Z Y,et al.Preparation and characterization of a potent, long-lasting recombinant human serum albumin-interferon-α2b fusion protein expressed in Pichia pastoris[J].Eur.J.Pharm.Biopharm,2007,67:301-308