重组人胰高糖素样肽-1突变体的串联体与人血清白蛋白融合蛋白的纯化及纯度鉴定
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摘要
本文利用构建好的重组Pichia Pastoris KM71/(GLP-1_(A2G))_2-HSA酵母工程菌株表达系统进行了发酵条件优化,并对目标蛋白进行了较深入的纯化工艺研究和简单的体内、体外活性研究。
将生产菌株增菌培养至一定菌体量后,用BMMY培养基悬浮菌体后定时添加甲醇进行诱导培养。研究表明重组Pichia Pastoris KM71/(GLP-1_(A2G))_2-HSA的最佳诱导条件为温度30℃、pH6.0、甲醇浓度2.0 %、诱导时间为60 h。此最优化条件既节省反应材料,又可以提高效率,目标蛋白的最高产量可达到210 mg/L。
发酵液经过4℃,10000rpm,15min离心除菌离心后,采用截留分子量为10KDa的Biomax超滤膜对发酵上清进行浓缩,浓缩倍数可达到20倍,回收率最高可达到90%。在精细分离过程中,首先将浓缩后的发酵液上Q-Seperose Fast Flow阴离子交换层析,上样后进行阶段洗脱,目标蛋白在盐浓度达到0.1mol/L时开始被洗脱下来,回收率达74%。所得到的洗脱成分处理后上HPLC检测,结果显示纯度为90%,SDS-PAGE显示为一条带。将Q-Seperose阴离子交换层析后的洗脱液浓缩后上Sephacryl S-200凝胶过滤,结果出现两个蛋白吸收峰,经检测后一个峰为目标蛋白分子吸收峰。将蛋白活性峰上HPLC检测,结果显示纯度达98%以上,经I125标记后,采用TCA沉淀法测定放射化学纯度达97%,纯度优于药代动力学研究的要求,达到了预期纯化目标。本纯化工艺由发酵上清经超滤、离子交换、凝胶过滤共3步组成,与文献报道的重组人血清白蛋白纯化工艺相比,具有流程简单、回收率高和成本较低等优点,为本产品的工业化生产奠定了基础。
融合蛋白rh(GLP-1_(A2G))_2-HSA在体外对胰岛原代细胞生长有较好的刺激作用,在浓度40 nmol/L时增殖率为35.4%,与单体的GLP-1有相似的增殖作用。
Intensive studies on the process of fermentation, purification and bioactivity were conducted in this thesis.
During the fermentation, the producing stain Pichia Pastoris KM71/(GLP-1_(A2G))_2-HSA was induced after 60 hours processing, then the concentration of (GLP-1_(A2G))_2-HSA in the fermentation liquid reached 210 mg/L. The optimized expression condition consisted temperature 30℃, pH 6.0 and the methanol induced concentration 2.0 %.
This research mainly dealt with purification of recombinant protein rh(GLP-1_(A2G))_2-HSA from fermentation broth. Highly purified rh(GLP-1_(A2G))_2-HSA was separated from fermentation broth by ultrafiltration concentration, ion exchange chromatography and gel filtration. The purified product was conformed as one single band by SDS-PAGE and its purity was identified as 98% by HPLC while its radioactive purity was identified as 97% by TCA deposition method after marked with 125I. The purity of rh(GLP-1_(A2G))_2-HSA met the requirement of drug activity and drug metabolism research. The total recovery yield reached 48.5 %. Highly purified rh(GLP-1_(A2G))_2-HSA could be gained by this purification method and layed a foundation for advanced drug activity and drug metabolism research.
Cell proliferative assay showed that the activity of rh(GLP-1_(A2G))_2-HSA remained well during the purification while the purified product had similar proliferative activity of isletβ-cell with GLP-1. When the concentration of rh(GLP-1_(A2G))_2-HSA is 40 nmol/L, the growth rate is 35.4%.
将生产菌株增菌培养至一定菌体量后,用BMMY培养基悬浮菌体后定时添加甲醇进行诱导培养。研究表明重组Pichia Pastoris KM71/(GLP-1_(A2G))_2-HSA的最佳诱导条件为温度30℃、pH6.0、甲醇浓度2.0 %、诱导时间为60 h。此最优化条件既节省反应材料,又可以提高效率,目标蛋白的最高产量可达到210 mg/L。
发酵液经过4℃,10000rpm,15min离心除菌离心后,采用截留分子量为10KDa的Biomax超滤膜对发酵上清进行浓缩,浓缩倍数可达到20倍,回收率最高可达到90%。在精细分离过程中,首先将浓缩后的发酵液上Q-Seperose Fast Flow阴离子交换层析,上样后进行阶段洗脱,目标蛋白在盐浓度达到0.1mol/L时开始被洗脱下来,回收率达74%。所得到的洗脱成分处理后上HPLC检测,结果显示纯度为90%,SDS-PAGE显示为一条带。将Q-Seperose阴离子交换层析后的洗脱液浓缩后上Sephacryl S-200凝胶过滤,结果出现两个蛋白吸收峰,经检测后一个峰为目标蛋白分子吸收峰。将蛋白活性峰上HPLC检测,结果显示纯度达98%以上,经I125标记后,采用TCA沉淀法测定放射化学纯度达97%,纯度优于药代动力学研究的要求,达到了预期纯化目标。本纯化工艺由发酵上清经超滤、离子交换、凝胶过滤共3步组成,与文献报道的重组人血清白蛋白纯化工艺相比,具有流程简单、回收率高和成本较低等优点,为本产品的工业化生产奠定了基础。
融合蛋白rh(GLP-1_(A2G))_2-HSA在体外对胰岛原代细胞生长有较好的刺激作用,在浓度40 nmol/L时增殖率为35.4%,与单体的GLP-1有相似的增殖作用。
Intensive studies on the process of fermentation, purification and bioactivity were conducted in this thesis.
During the fermentation, the producing stain Pichia Pastoris KM71/(GLP-1_(A2G))_2-HSA was induced after 60 hours processing, then the concentration of (GLP-1_(A2G))_2-HSA in the fermentation liquid reached 210 mg/L. The optimized expression condition consisted temperature 30℃, pH 6.0 and the methanol induced concentration 2.0 %.
This research mainly dealt with purification of recombinant protein rh(GLP-1_(A2G))_2-HSA from fermentation broth. Highly purified rh(GLP-1_(A2G))_2-HSA was separated from fermentation broth by ultrafiltration concentration, ion exchange chromatography and gel filtration. The purified product was conformed as one single band by SDS-PAGE and its purity was identified as 98% by HPLC while its radioactive purity was identified as 97% by TCA deposition method after marked with 125I. The purity of rh(GLP-1_(A2G))_2-HSA met the requirement of drug activity and drug metabolism research. The total recovery yield reached 48.5 %. Highly purified rh(GLP-1_(A2G))_2-HSA could be gained by this purification method and layed a foundation for advanced drug activity and drug metabolism research.
Cell proliferative assay showed that the activity of rh(GLP-1_(A2G))_2-HSA remained well during the purification while the purified product had similar proliferative activity of isletβ-cell with GLP-1. When the concentration of rh(GLP-1_(A2G))_2-HSA is 40 nmol/L, the growth rate is 35.4%.
引文
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8. Drucker DJ, Biological actions and therapeutic potential of the glucagons-like peptides.Gastroenterology, 2002,122(2):531-544
9.冯波,李传琼.2型糖尿病血胰淀素、胰升血糖素肽水平及胰岛功能变化的探讨[J].内分泌代谢杂志,1998,14(1):44-45
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11. 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. Diabetes Metab Res Rev, 2005, 21(2):313-331
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19. Pospisilik J A, Stafford S G, Demuth HU, 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.Diabetes,2002,51(4):943-950
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29.吴诚,钟延强.干扰素缓释给药系统的研究进展[J].药学实践杂志, 2003, 21(2):87-89
30. Kajihara M, Sugie T, Mizuno M, et al. Development of new drug delivery system for protein drugs using silicone. Journal of Controlled Release, 2000, 12(1):49-61
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32.张华金,李娜,杜学志.干扰素的剂型及临床应用[J].华西药学杂志,2004,19(1):53-55
33.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
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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.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
4.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
5. Nauck M A, Niedreichholz 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(5Ptl):E981-988
6. Radosavljevic T, Todorovic V, Sikic B, Role of glucagon-like peptide 1(GLP-1) in regulation of insulin secretion.Srp Arh Celok Lek, 2002,130(11-12):416-419
7. Holst JJ,Glucagon-like peptide-1,a gastrointestinal hormone with a pharmaceutical potential.Curr Med Chem,1999,6(11):1005-1017
8. Drucker DJ, Biological actions and therapeutic potential of the glucagons-like peptides.Gastroenterology, 2002,122(2):531-544
9.冯波,李传琼.2型糖尿病血胰淀素、胰升血糖素肽水平及胰岛功能变化的探讨[J].内分泌代谢杂志,1998,14(1):44-45
10. Hoist JJ. Glucagon-like peptide 1(GLP-1):an intestinal hormone, signaling nutritional abundance, with an unusual therapeutic potential. Trends Endocrinal Metab, 1999,10(6):229-235
11. 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. Diabetes Metab Res Rev, 2005, 21(2):313-331
12. Drucker D J. Minireview: the glucagon-like peptide. Endocrinology,2001,142(2): 521-527
13. Deacon C F, Knudsen L B, Madson K, et al. Dipeptidyl-peptidaseⅣresistant analogues of glucagons-like peptide-1which have extended metabolic stability and improved biological activity. Diabetologia,1998,41(3):271-278
14.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
15. Kreymann B, Williams G, Ghatei MA, et al. Glucagon-like peptide-1 7-36: a physiological incretin in man Lancet. 1987;2(8571):1300-1303
16. 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. Clin. Endocrinol. Metab.2003,88(4):1772-1779
17. Turton M D, O'Shea D, Gunn I, et al. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature,1996,379(6560):69-72
18. Ahrén B, Winzell M S, Wierup N, et al. Hughes, 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.Regul.Pept.2007, 143(3) :97-103
19. Pospisilik J A, Stafford S G, Demuth HU, 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.Diabetes,2002,51(4):943-950
20.姚艳丽,冯凭.胰高血糖素样肽-1与1型糖尿病治疗[J].生命的化学,2005,25(4)316-317
21.郭徽,张胜兰.胰高血糖素样肽-1及其对糖尿病的治疗研究进展[J].实用医学杂志,2005,22(6):545-546
22. Wang Q, Li L, Xu E, et al. Glucagon-like peptide 1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells. Diabetologia, 2004,47(3):478-487
23. Behme MT. Glucagon-like peptide I improved glycemic control in type I diabetes BMC Endocr Disord.Diabetes,2003,3(1):3-8
24. Parkes D G, Pirrner R, Jodka C, et al. Insulinotropic action of exendin-4 and glucagon-like peptide-1 in vivo and in vitro. Metab,2001,50(5):583-589
25. Toft Nielsen M B, Madsbad S, Holstj J. Determinants of the offectiveness of glucagon-like peptide-1 in type 2 diabetes. J Clin Endocrinol Metab, 2001,86(8):3853-3860
26. Conarello SL, Li Z, Ronan J, et al. Mice lacking dipetidyl peptidaseⅣare protected against obesity and insulin resistance. Proc Natl Acad Sci USA, 2003, 100(11):68-75
27. Mentlein R. Dipeptidy-peptidaseⅣ(CD26):role in the inactivation of regulatory peptides.Regul Pept, 1999,85(6):9-24
28.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):23-27
29.吴诚,钟延强.干扰素缓释给药系统的研究进展[J].药学实践杂志, 2003, 21(2):87-89
30. Kajihara M, Sugie T, Mizuno M, et al. Development of new drug delivery system for protein drugs using silicone. Journal of Controlled Release, 2000, 12(1):49-61
31.罗东.干扰素剂型研究进展[J].中国新医药, 2003, 2(9):49-51
32.张华金,李娜,杜学志.干扰素的剂型及临床应用[J].华西药学杂志,2004,19(1):53-55
33.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
34. Poznansky MJ, Halford J, Taylor D. Growth hormone-albumin conjugates Reduced renal toxicity and altered plasma clearance. FEBS letters, 1988, 239(1):18-22
35. Khanna N.A. Human Genome Sciences Reports Positive Results of Phase 1/2 Clinical Trial ofAlbuferon in Chronic HepatitisC. HGSI Press,2004,11(2):2-8
36. Brown J.R.Albumin Structure, unction and Uses.Oxford: pergamon Press,1997.27-51
37. Behrens.Large fragments of human serum albumin. Bio Chem, 1977,161(3):619-625
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