人胰岛素样生长因子1（hIGF-1）中试工艺的研究
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摘要
本研究用改进的RT-PCR法合成人肝脏cDNA,构建出cDNA文库,并从中分离出人胰岛素样生长因子1(human insulin like growth factor 1,hIGF-1)基因。将hIGF-1基因酶切后插入到pGEX-1λT载体中,连接转化大肠杆菌NM522,筛选克隆,酶切鉴定后测序,并进行了表达。将IPTG诱导表达的菌体离心收集,取少量菌体进行SDS-PAGE分析,在分子量34KD处可见明显高表达带(对照菌没有表达),Western印迹表明重组蛋白具有hIGF-1的抗原性。
在完成以上研究的基础上,鉴于原核分泌型表达技术的优势,我们根据原核细胞分泌表达机制及需要,重新设计引物,利用PCR法分别分离出碱磷酸酶启动子(phoA),STII信号肽及hIGF-1编码基因,将它们连接到pUC载体,测序后,通过点突变技术对全基因序列进行了校正。
分别构建出两套分泌型表达载体,并将其命名为pCSA和pCST,随后将上述基因装入到pCSA和pCST中,转化大肠杆菌W3110,得到工程菌pCSA/IGF-1/W3110,pCST/IGF-1/W3110,并进行了表达。经SDS-PAGE检测,在分子量7.6KD处有明显高表达带,Western印迹表明重组蛋白具有hIGF-1的抗原性。
根据中国生物制品检定规程的有关要求,我们进行了基因工程大肠杆菌工程菌的筛选和鉴定,得到工程菌株pCSA/IGF-1/W3110 ,pCST/IGF-1/W3110,经传代培养,提质粒酶切鉴定及蛋白表达测定表明,经100次传代后质粒不丢失,酶切鉴定表明为阳性克隆,蛋白表达稳定。
根据中试工艺的要求,我们初步建立了分泌型hIGF-1的生产工艺,即为;种子液制备→发酵培养→离心收集菌体深度冷冻→rhIGF-1低渗震荡溶出→层析前处理→疏水层析→分子筛→离子交换→半成品。参照有关文献,以NIH3T3作为靶细胞,用XTT掺入法,初步建立了hIGF-1生物活性检定方法。
Human insulin-like growth factor I (hIGF-1) is a kind of multipotential regulatory factor secreted by cell, and has wide biological effects. It can promote proliferation and differentiation of many kind of cell. As a result human IGF-1 (hIGF-1) has been put in use to treat many diseases such as diabetes, insulin-resistant syndrome, dwarfism, some nervous system deseases and other obstinate diseases. The clinic outcomes were rather encounging.
Unfortunately the quantity of hIGF-1 in blood is very limited, moreover it mainl exists in the form of inactive complex. In order to get sufficient quantities of pure hIGF-1 with bioactivity to meet the demand of basic research and clinical uses, We first builded the human liver tissue cDNA library and from it gene fragment of hIGF-1 was isolated. After DNA sequencing result proved to be the right sequence, It was cloned into vector pGEX-1λT so that the fusion expressing vector pGEX/hIGF-1 was constructed. When it was transformed into E.coli NM522 the fusion protein was expressed. The protein was indicated hIGF-1 antigenicity by analysis of SDS-PAGE and Western blot.
At the same time, we construct two prokaryotic secretory expressing vector pCSA and pCST according to the principle of prokaryotic secretory expression. First, promotor alkaline phosphatase (phoA), signal peptide STII and hIGF-1 gene were obtained by PCR, and then they were inserted into vector pUC. After the whole gene sequence was corrected by technology of point mutation, it was inserted into vector pCSA and pCST, which came to the two secretory expressing prokaryotic vector of pCSA and pCST. After it was transformed to E.coli W3110, two engineering stains of pCSA/IGF-1/W3110 and pCST/IGF-1/W3110 were obtained to express corresponding protein. The protein expressed detected by SDS-PAGE showed a high expressed protein band with a relative molecular weight of 7.6KD, western blot showed the hIGF-1 antigenicity.
Then Stability of the engineering strain and the method for its screening and preservation were studied, as well as the semi-works technology of secretory expressed hIGF-1 and detective method of its bioactivity.
In the end, the fermentation, purification technology on secretory expression of hIGF-1 was primarily set up, as well as the detective method of the bioactivity of hIGF-1. The construction of semi-works technology of secretory-expressed hIGF-1 and the establishment of detective method of hIGF-1 bioactivity will play an elementary role of further study on the function and characteristics of hIGF-1, as well as the industrialization of hIGF-1.
在完成以上研究的基础上,鉴于原核分泌型表达技术的优势,我们根据原核细胞分泌表达机制及需要,重新设计引物,利用PCR法分别分离出碱磷酸酶启动子(phoA),STII信号肽及hIGF-1编码基因,将它们连接到pUC载体,测序后,通过点突变技术对全基因序列进行了校正。
分别构建出两套分泌型表达载体,并将其命名为pCSA和pCST,随后将上述基因装入到pCSA和pCST中,转化大肠杆菌W3110,得到工程菌pCSA/IGF-1/W3110,pCST/IGF-1/W3110,并进行了表达。经SDS-PAGE检测,在分子量7.6KD处有明显高表达带,Western印迹表明重组蛋白具有hIGF-1的抗原性。
根据中国生物制品检定规程的有关要求,我们进行了基因工程大肠杆菌工程菌的筛选和鉴定,得到工程菌株pCSA/IGF-1/W3110 ,pCST/IGF-1/W3110,经传代培养,提质粒酶切鉴定及蛋白表达测定表明,经100次传代后质粒不丢失,酶切鉴定表明为阳性克隆,蛋白表达稳定。
根据中试工艺的要求,我们初步建立了分泌型hIGF-1的生产工艺,即为;种子液制备→发酵培养→离心收集菌体深度冷冻→rhIGF-1低渗震荡溶出→层析前处理→疏水层析→分子筛→离子交换→半成品。参照有关文献,以NIH3T3作为靶细胞,用XTT掺入法,初步建立了hIGF-1生物活性检定方法。
Human insulin-like growth factor I (hIGF-1) is a kind of multipotential regulatory factor secreted by cell, and has wide biological effects. It can promote proliferation and differentiation of many kind of cell. As a result human IGF-1 (hIGF-1) has been put in use to treat many diseases such as diabetes, insulin-resistant syndrome, dwarfism, some nervous system deseases and other obstinate diseases. The clinic outcomes were rather encounging.
Unfortunately the quantity of hIGF-1 in blood is very limited, moreover it mainl exists in the form of inactive complex. In order to get sufficient quantities of pure hIGF-1 with bioactivity to meet the demand of basic research and clinical uses, We first builded the human liver tissue cDNA library and from it gene fragment of hIGF-1 was isolated. After DNA sequencing result proved to be the right sequence, It was cloned into vector pGEX-1λT so that the fusion expressing vector pGEX/hIGF-1 was constructed. When it was transformed into E.coli NM522 the fusion protein was expressed. The protein was indicated hIGF-1 antigenicity by analysis of SDS-PAGE and Western blot.
At the same time, we construct two prokaryotic secretory expressing vector pCSA and pCST according to the principle of prokaryotic secretory expression. First, promotor alkaline phosphatase (phoA), signal peptide STII and hIGF-1 gene were obtained by PCR, and then they were inserted into vector pUC. After the whole gene sequence was corrected by technology of point mutation, it was inserted into vector pCSA and pCST, which came to the two secretory expressing prokaryotic vector of pCSA and pCST. After it was transformed to E.coli W3110, two engineering stains of pCSA/IGF-1/W3110 and pCST/IGF-1/W3110 were obtained to express corresponding protein. The protein expressed detected by SDS-PAGE showed a high expressed protein band with a relative molecular weight of 7.6KD, western blot showed the hIGF-1 antigenicity.
Then Stability of the engineering strain and the method for its screening and preservation were studied, as well as the semi-works technology of secretory expressed hIGF-1 and detective method of its bioactivity.
In the end, the fermentation, purification technology on secretory expression of hIGF-1 was primarily set up, as well as the detective method of the bioactivity of hIGF-1. The construction of semi-works technology of secretory-expressed hIGF-1 and the establishment of detective method of hIGF-1 bioactivity will play an elementary role of further study on the function and characteristics of hIGF-1, as well as the industrialization of hIGF-1.
引文
1. Ellis S, Huble J, Simpson ME. Influence of hypophysectomy and growth hormone on cartilage sulfate metabolism[J]. Proc Soc Exp Biol Med, 1953, 84:603~605.
2. Salmon Jr WD, Daughaday WH. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro[J]. J Lab Clin Med, 1957, 49:825~836.
3. Daughaday WH, Hall K. Somatomedin: a proposed designation for the sulfationf actor[J]. Nature, 1972, 235:107.
4. Jones JR, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocrine Reviews, 1995, 16:3~34.
5. Froesch ER, Burgi H. Antibody-suppressible and nonsuppressible insulin-like activities in human serum and their physiologic significance. An insulin assay with adipose tissue of increased precision and specificity[J]. J Clin Invest, 1963, 42:1816-1834.
6. Rinderknecht E, Humbel RE. Polypeptides with nonsuppressible insulin-like and cell-growth promoting activities inhuman serum: isolation, chemical characterization, and some biological properties of forms 1 and 2[J]. Proc Natl Acad Sci, 1976, 73:2365-2369.
7. Rinderknecht E, Hmbel RE. Primary structure of human insulin-like growth factor 2[J]. FEBS Lett, 1978, 89:283-286.
8. Rinderknecht E, Humbel RE. The amino acid sequence of human insulin-like growth factor 1 and its structural homology with proinsulin[J]. J Biol Chem, 1978, 253: 2769-2776.
9. Daughaday WH, Hall K, Salmon Jr WD. On the nomenclature of the somatomedins and insulin-like growth factor [J]. J Clin Endocrinol Metab, 1987, 65: 1075.
10. 刘宝英,王会信. 胰岛素样生长因子研究进展[J]. 国外医学分子生物学分册,1996,16(3):103-108.
11. Rene E ,Humbel RE. Review insulin-like growth factors 1 and 2[J]. J Biochem, 1990, 190:445-462.
12. Sara VR, Carlsson-Skwirut C, Andersson C et al. Characterization of somatomedins from human fetal brain: identification of a variant form of insulin-like growth factor1 [J]. Proc Natl Acada Sci USA, 1986, 83(13):4904-4907.
13. Carlsson-Skwirut C, Jornvall H, Holmgren A et al.Isolation and characterization of variant HHIGF-1 as wall as IGF-2 from adult human brain[J]. FEBS Lett, 1986, 201(1): 46-50.
14. Ogasawara M, Karey KP, Marquardt H et al. Identification and purification of truncated insulin-like growth factor1from porcine uterus[J]. Biochemistry, 1989, 28(6):2710-2721.
15. Francis GL, Upton FM, Ballard FJ et al. Insulin-like growth factors1 and 2 in bovine colostrums. Sequences and biological activities compared with those of a potent truncated form[J]. Biochem J, 1988, 251(1): 95-103.
16. Carlsson-Skwirut C, Lake M, Hartmanis M et al. A comparison of the biological activity of the recombinant intact and truncated insulin-like growth factor1(HHIGF-1)[J]. Biochim Biophys Acta, 1989, 1011:192-197.
17. Robert MC, Timothy SH, Iain DC. Solution structure of human insulin-like growth factor 1: a nuclear magnetic resonance and restrained molecular dynamics stucy[J]. Biochemistry, 1991, 30(22): 5484-5491.
18. Akihiro S, Shigenori N, Tadayasu O et al. 1H-NMR assignment and secondary structure of human insulin-like growth factor-1(IGF1)in solution[J]. J Biochem, 1992, 111(4):529-536.
19. Van den Brande JL. Structure of human insulin-like growth factors: relationship to function. In: Paul N eds. The insulin-like growth factors[J].New York: Oxford university press, 1992.12-44.
20. Rosen CJ, Pollak M. Circulating HHIGF-1: New perspectives for a new century[J]. TEM, 1999, 10:1362141.
21. Twigg SM, Baxter RC. IGF binding protein 5 forms an alternative ternary complex with IGFs and the acid lable subunit[J]. J Biol Chem, 1998, 273:607426079.
22. Johansson AG, Lindh E, Ljunghall S. IGFs: function and clinical importance 4 growth hormone, insulin-like growth factors 1, and bone: a clinical review[J]. J Intl Med, 1993, 234:553-560.
23. Zapf J, Walter H, Froesch ERJ. Radioimmunological determination of insulin-like growth factors 1and 2 in normal subjects and in patients with growth disorders and extrapancreatic tumor hypoglycemia[J]. J Clin Invest, 1981, 68(5): 1321-1330.
24. Cohick WA, Clemmons DR. The insulin-like growth factors[J]. Annu Rev Physiol, 1993, 55:131-153.
25. 李丽艳, 路新国. 胰岛素样生长因子系统与运动系统的关系研究进展[J]. 中国康复医学杂志, 2005, 20(8):632-635.
26. 陈照丽,王欣,黄秉人. IGF 系统的生物学功能及其与肿瘤的关系 [J]. 国外医学分子生物学分册,2003,25(1):34-37.
27. 刘宝英,王会信. 胰岛素样生长因子 1 的结构与功能研究进展[J]. 生物化学与生物物理进展,1998,25(4):311-315.
28.Akihiro S, Shigenori N, Tadayasu O et al. Three-dimensional structure of human insulin-like growth factor-1(IGF1)detemined by 1H-NMR and distance geometry[J]. Int J Peptide Protein Res, 1993, 41(5):433-440.
29. Bayne ML, Applebaum J, Chicchi CG et al. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and type 2 insulin-like growth factor receptor[J]. J Biol Chem, 1988, 263(13):6233-6239.
30. Cascieri MA, Chicchi GC, Applebaum J et al. Structural analogs of human insulin-like growth factor(IGF) 1 with altered affinity for type 2 IGF receptor[J]. J Biol Chem, 1989, 264(4):2199-2202.
31. Morgan DO, Edman JC, Standring DN et al. Insulin-like growth factor 2 receptor as a multifunctional bindig protein[J]. Nature, 1987, 329(24):301-307.
32. Rajaram S, Baylink DJ, Mohan S. Insulin-like growth factor binding proteins in serum and other biological fluids: regulation and functions[J]. Endocr Rev, 1997, 18:801-831.
33. 高玉红,李庆章. 胰岛素生长因子系统研究进展[J]. 中国乳品工业,2006, 34(4):46-49.
34. Victor KMH, Douglas GM, Patric JDD et al. IGF-binding protein mRNAs in the human fetus, Tissue and cellular distribution of developmental expression[J]. Human Res, 1996, 45(1):160-166.
35. Clemmons DR, Moses AC, Mckay MJ,etal. The combination of insulin-like growth factor 1 and insulin-like growth factor bindingprotein-3 reduces insulin requirements in insulin-dependent type 1 diabetes: evidence for invivo biological activity[J]. J Clin Endocrinol Metab, 2000, 85:1518-1524.
36. 段宇,刘超,汪承亚,等. 胰岛素样生长因子 1 研究进展[J]. 国外医学内分泌学分册,2001,21(6):305-307.
37. Rosen CJ, Conover C. GH/IGF-1 axis in aging: a summary of a NIA-sponsored symposium[J]. J Clin Endocrinol Metab, 1997, 82:3919-3922.
38. Estivariz CF, Zieyler TR. Nutrition and the insulin-like growth factor system. Endocrine, 1997, 7:65-71.
39. Dunger DB, Cheetham TD. Growth hormone insulin-like growth factor I axis in insulin-dependent diabetes mellitus. Horm Res, 1996, 46:2-6.
40. Harrela M, Kao PC, Matheny AP, et al. Genetic and environmentalcomponents of inter-individual variation in circulating levels of IGF-1, IGF-2, IGFBP-1, IGFBP-3[J]. J Clin Invest, 1996, 98:2612-2615.
41. Rosen CJ, Conover C. Circulating and skeletal IGF-1 concentrations in two inbred strains of mice with different bone densities[J]. Bone, 1997, 21:217-223.
42. Rosen CJ, Kurland ES, Vereauit D, et al. Association between serum IGF-1 and a simple sequence repeat in the IGF-1 gene: implications for genetic studies of BMD[J]. J Clin Endocrinol Metab, 1998, 83:228622290.
43. Neuberg M, Buckbinder L, Seizinger B, et al. The P53/IGF-1 receptor axis in the regulation of programmed cell death[J]. Endocrine, 1997, 138:1072109.
44. 杜惠峰, 傅中滇. 糖尿病及其国内流行现状[J]. 生物学教,2002,27(1):4-5.
45. 王克安. 中国糖尿病的防治和研究[J]. 中华流行病学杂志,1999,20(5):260-263.
46. Alan DC. 糖尿病:过去、现在和将来[J]. 国外医学内分泌学分册,2005, 25(5):附录 3-5.
47. 韩炳鑫,李晓东,刘小虎,等. 第 65 届美国糖尿病年会纪要[J]. 中国全科医学,2006,9(16):1385-1386.
48. Cusz K, Defronzo RA. Treatment of NIDDM, IDDM and other insulin-resistent states with IGF-1[J]. Diabetes Rev, 1998, 3:206-229.
49. Dunger DB, Cheetham TD. Holly JMP. Does recombinant insulin-like growth factor I have a role in the treatment of IDDM during adolescence[J]. Acta Paediatr, 1993, 338(Suppl): 49-52.
50. 许辉,蒙碧辉. IGF-1 在Ⅰ型糖尿病中的研究进展[J]. 医学研究杂志,2006,35(12):79-81.
51. Clauson PG, Brismar K, Hall K, et al. Insulin-like growth factor-I andinsulin-like growth factor binding protein-1 in a representative population of type 2 diabetic patients in Sweden [J], Scand J Clin Lab Invest, 1998, 58:353-360.
52. 张金梅,张洁,刘金玲,等. 胰岛素样生长因子 1 与糖尿病及其并发症的关系[J], 中西医结合心脑血管病杂志,2007,5(1):58-59.
53. Sasaoka T, Kobayashi M. Takata Y, et al. Clarification of signaling pathways mediated by insulin and insulin-like growth factor I receptors in fibroblasts from patients with specific defect in insulin receptor [J]. Diabetes, 1998, 37:1515-1523.
54. Kato M. Biological and clinical analyses of the mechanism of growth retardation and the effect of recombinant human insulin-like growth factor-1(rhIGF-1) treatment on glucose metabolism and growth in leprechaunism with severe insulin resistance diabetes [J]. Hokkaido Igaku Zasshi, 1998, 73:613-625.
55. Laron Z, Anin S, Klipper-Aurbach Y, et al. Effects of insulin-like growth factors on linear growth, head circumference, and body fat in patients with Laron-type dwarfism[J]. Lancet, 1992, 339:1258-1261.
56. Brain CE, Hubbard M, Preece MA, et al. Metabolic states of children with growth hormone insensitivity syndrome and responses to treatment with HHIGF-1 [J]. Horm Res, 1998, 50:61-70.
57. Jehle P, Schulten K, Schulz W, et al. Serum levels of insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 to -6 and their relationship to bone metabolism in osteoporosis patients. Eur J Intern Med, 2003, 14:32238.
58. Reinecke M, Schmid AC, Hunziker EB, et al. Effect of growth hormone and insulin-like growth factor I (IGF-1) on the expression of IGF-1 messenger ribonucleic acid and peptide in rat tibial growth plate and articular chondrocytes in vivo. Endocrinology, 2000, 141:2847-2853.
59. Miyatake N Shikata K, Wada J, et al. Differential distribution of insulin-like growth factor-1 and insulin-like growth factor binding proteins in experimental diabetic rat kidney [J]. Nephron, 1999, 81:317-323.
60. Gambaro G, Baggio B. Growth factors and the kidney in diabetes mellitus[J]. Crit Rev Clin Lab Sci. 1998, 35:117-151.
61. Herndon DN, Ramzy PI, Debroy MA, et al. Muscle protein catabolism after severe burn: effects of IGF-1/IGFBP-3 treatment[J]. Ann Surg. 1999, 229:713-722.
62. Mauras N, O’Brien KO, Welch S, et al. Insulin-like growth factor I and growth hormone(GH) treatment in GH-deficient humans: differential effects on protein, glucose, lipid, and calcium metabolism[J].J Clin Endocrinol Metab 2000, 85:1686-1694.
63. Ishii DN. Implication of insulin-like growth factors in the pathogenesis of diabetic neuropathy[J]. Brain Res Revi, 1995, 20:47267.
64. Torresaleman I, Barrios V, Berciano J. The peripheral insulin-like growth factor system in amyotrophic lateral sclerosis and in multiple sclerosis[J]. Neurology, 1998, 50: 772-776.
65. 徐伟文,吴岚晓, 李明. 胰岛素样生长因子-1 的临床应用及其前景[J]. 国外医学内分泌学分册,1999,19(6):270-273.
66. Kovacs GT. Worgall S, Schwalbach P, et al. Hypoglycemic effects of insulin-like growth factor-1 in experimental uremia: can concomitant growth hormone administration prevent this effect [J]. Horm Res, 1999,51:193-200.
67. Roith DL. Insulin-like growth factors[J]. N Engl J Med, 1997, 336: 633-640.
68. Acerini CL, Patton CM, Savage MO, et al. Randomised placebo-controlled trial of human recombinant insulin-like growth factor-1 plus intensive insulin therapy in adolescents with insulin-dependent diabetes mellitus[J]. Lancet, 1997, 350:1199-1204.
69. Kuroki M, Kawakami MD. Diabetic retinopathy-the mechanisms of the ocular neovascularization and the development of antiangiogenic drugs [J]: Nippon Rinsho, 1999. 57:584-589.
70. http://www.d1.com.cn/html/01101231.htm
71. 黄胜炎. 国外新批准上市的药物新制剂新剂型[J]. 中国制药信息,2006,22(4):23-26.
72. www.mofcom.gov.cn/aarticle/o/di/200603/20060301743202.html
73. 陆志城. 医药经济报,2006 年医院周刊第 21-23 期.
74. Lewis ME, Neff NT, Contreras PC et al. Insulin-like growth faetor I: Potential for treatment of motor neuronal disorders[J]. Exptl Neurol, 1993, 124:73-88.
75. 聂尚海、刘宝英. 胰岛素样生长因子 I 与肌萎缩性侧索硬化症[J].神经生化学通讯,1998,11(3,4):77-81.
76. Fervenza. Insulin-like growth factor-I treatment to enhance renal function in advanced chronic renal failure [J], Ren Fail,1998,20:349-354.
77. Yarwood. Administration of human recombinant insulin-like growth factor-I in critically ill patients [J].Crit Care Med, 1997, 25:1352-1356.
78. 李伯良、胡明、杨新颖等. 人胰岛素生长因子基因人工合成、克隆及其表达[J]. 生物化学与生物物理学报, 1994,26(2):153-160.
79. 李军、李伯良. 蛋白 A 信号肽引导的 E.coli 外分泌高表达异源蛋白[J].生物化学与生物物理学报,1995,27(6)616-623.
80. 刘宝英、赵明、王会信等. 人胰岛素样生长因子 I 在大肠杆菌中的表达研究[J]. 中国生物化学与分子生物学报, 1998,14(1):47-51.
81、刘宝英、聂尚海、杜清友等. 人胰岛素样生长因子 I 突变体的构建、表达及其鉴定[J]. 军事医学科学院院刊,1999,23(2):94-96.
82. 梁东春,左爱军,吴亚锋,等. 重组人胰岛素样生长因子 I 的制备[J]. 天津医药, 2005,33(7):434-436.
83. 凌明圣、许祥裕、施凤霞等. 人胰岛素样生长因子 I 基因的半合成及克隆表达[J]. 中国生化药物杂志, 1999,20(4):166-169.
84. 吴岚晓,李 明,王 萍,等. 人胰岛素样生长因子-1 在大肠杆菌中的表达[J]. 药物生物技术,2001,8(4):185-188.
85. 吴岚晓,李明,陈白虹,等. 重组人胰岛素样生长因子-1 分离纯化工艺的建立[J]. 中国药学杂志,2002,37(5):376-379.
86. 高媛,余榕捷,洪岸,等. 人类胰岛素生长因子基因的合成、表达与纯化[J]. 中国病理生理杂志,2005,21(2):260-265.
87. 刘侃,汪炬,谢秋玲,等. 重组类胰岛素样生长因子-1 的纯化与复性 [J]. 中国生物工程杂志, 2006,26(2):29-33.
88. 魏辉俊、黄秉仁、蔡良琬. 类胰岛素生长因子 I 的克隆、表达、分离纯化与活性检测[J]. 中国医学科学院学报, 1997,19(5):342-345.
89. 赖心田,周鹏,洪葵. 人胰岛素样生长因子-1 在毕赤酵母中的表达研究 [J].药物生物技术,2002,9(3):133-136.
90. 刘晓辉、梁述文、王伟. 截短型人胰岛素样生长因子 rIGF-1 在大肠杆菌中的表达,纯化和鉴定[J]. 中国生物化学与分子生物学报,1999,15(5):738-741.
91. 李莹、邓鸿业、狄春辉等. 重组人截短型胰岛素样生长因子 I 在大肠杆菌中的高效表达[J]. 中国免疫学杂志, 2000,16(2):57-60.
92. 周鹏,王跃进,贺普超,等. 人胰岛素样生长因子-1 基因农杆菌工程菌株的构建[J]. 西北农林科技大学学报,2001,29(3):19-23.
93. 张英华,王小佳. 用于转化甘蓝的胰岛素样生长因子 I 的合成及MAR 调控的表达载体构建[J]. 农业生物技术学报,2004,12(6):731-732.
94. 赵红、 汪承亚、陈华群,等. 昆虫细胞表达重组人胰岛素样生长因子 I 的研究[J]. 南京医科大学学报,2000,20(4):243-245.
95. 段 宇,汪承亚,赵 红,等. 利用杆状病毒载体在家蚕幼虫中高效表达人胰岛素样生长因子-1 的研究[J]. 中国生化药物杂志,2001,22(6):274-277.
96. 徐志伟,娄亚欣,李 莹,等. 人胰岛素样生长因子 1 的真核细胞表达及其鉴定[J]. 免疫学杂志,2001,17(1):1-3.
97. 岳凤鸣,赵焕英,杨慧,等. 人胰岛素样生长因子-1 基因的克隆、表达和活性检测[J]. 解剖学报,2003,34(3):306-311.
98. 张绍昆,刘 一,单玉兴,等. 人胰岛素样生长因子-1 稳定表达细胞株的构建及鉴定[J].吉林大学学报 (医学版) ,2003,29(1):41-43.
99. 张海宁,侯筱魁,杨冠珍,等. 人 IGF-1 基因真核表达质粒pIRES2-EGFP- IGF-1 的构建[J]. 上海第二医科大学学报,2004,24(5):359-361.
100. 祁雅慧,王雅梅,孙丽翠,等. 人胰岛素样生长因子-1 基因的克隆、表达及生物活性分析[J]. 首都医科大学学报,2004,25(2):154-158.
101. 张鹏,陈世益,陈疾忤,等. 人胰岛素样生长因子-1 真核表达质粒的构建及其在成肌细胞中的表达 [J].中国运动医学杂志,2005,24(1):9-20.
102. 周海斌,郑祖根,董启榕. 人胰岛素样生长因子-1 的真核表达及其生物学功能研究[J].中国病理生理杂志,2007,23(1):27-29.
2. Salmon Jr WD, Daughaday WH. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro[J]. J Lab Clin Med, 1957, 49:825~836.
3. Daughaday WH, Hall K. Somatomedin: a proposed designation for the sulfationf actor[J]. Nature, 1972, 235:107.
4. Jones JR, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocrine Reviews, 1995, 16:3~34.
5. Froesch ER, Burgi H. Antibody-suppressible and nonsuppressible insulin-like activities in human serum and their physiologic significance. An insulin assay with adipose tissue of increased precision and specificity[J]. J Clin Invest, 1963, 42:1816-1834.
6. Rinderknecht E, Humbel RE. Polypeptides with nonsuppressible insulin-like and cell-growth promoting activities inhuman serum: isolation, chemical characterization, and some biological properties of forms 1 and 2[J]. Proc Natl Acad Sci, 1976, 73:2365-2369.
7. Rinderknecht E, Hmbel RE. Primary structure of human insulin-like growth factor 2[J]. FEBS Lett, 1978, 89:283-286.
8. Rinderknecht E, Humbel RE. The amino acid sequence of human insulin-like growth factor 1 and its structural homology with proinsulin[J]. J Biol Chem, 1978, 253: 2769-2776.
9. Daughaday WH, Hall K, Salmon Jr WD. On the nomenclature of the somatomedins and insulin-like growth factor [J]. J Clin Endocrinol Metab, 1987, 65: 1075.
10. 刘宝英,王会信. 胰岛素样生长因子研究进展[J]. 国外医学分子生物学分册,1996,16(3):103-108.
11. Rene E ,Humbel RE. Review insulin-like growth factors 1 and 2[J]. J Biochem, 1990, 190:445-462.
12. Sara VR, Carlsson-Skwirut C, Andersson C et al. Characterization of somatomedins from human fetal brain: identification of a variant form of insulin-like growth factor1 [J]. Proc Natl Acada Sci USA, 1986, 83(13):4904-4907.
13. Carlsson-Skwirut C, Jornvall H, Holmgren A et al.Isolation and characterization of variant HHIGF-1 as wall as IGF-2 from adult human brain[J]. FEBS Lett, 1986, 201(1): 46-50.
14. Ogasawara M, Karey KP, Marquardt H et al. Identification and purification of truncated insulin-like growth factor1from porcine uterus[J]. Biochemistry, 1989, 28(6):2710-2721.
15. Francis GL, Upton FM, Ballard FJ et al. Insulin-like growth factors1 and 2 in bovine colostrums. Sequences and biological activities compared with those of a potent truncated form[J]. Biochem J, 1988, 251(1): 95-103.
16. Carlsson-Skwirut C, Lake M, Hartmanis M et al. A comparison of the biological activity of the recombinant intact and truncated insulin-like growth factor1(HHIGF-1)[J]. Biochim Biophys Acta, 1989, 1011:192-197.
17. Robert MC, Timothy SH, Iain DC. Solution structure of human insulin-like growth factor 1: a nuclear magnetic resonance and restrained molecular dynamics stucy[J]. Biochemistry, 1991, 30(22): 5484-5491.
18. Akihiro S, Shigenori N, Tadayasu O et al. 1H-NMR assignment and secondary structure of human insulin-like growth factor-1(IGF1)in solution[J]. J Biochem, 1992, 111(4):529-536.
19. Van den Brande JL. Structure of human insulin-like growth factors: relationship to function. In: Paul N eds. The insulin-like growth factors[J].New York: Oxford university press, 1992.12-44.
20. Rosen CJ, Pollak M. Circulating HHIGF-1: New perspectives for a new century[J]. TEM, 1999, 10:1362141.
21. Twigg SM, Baxter RC. IGF binding protein 5 forms an alternative ternary complex with IGFs and the acid lable subunit[J]. J Biol Chem, 1998, 273:607426079.
22. Johansson AG, Lindh E, Ljunghall S. IGFs: function and clinical importance 4 growth hormone, insulin-like growth factors 1, and bone: a clinical review[J]. J Intl Med, 1993, 234:553-560.
23. Zapf J, Walter H, Froesch ERJ. Radioimmunological determination of insulin-like growth factors 1and 2 in normal subjects and in patients with growth disorders and extrapancreatic tumor hypoglycemia[J]. J Clin Invest, 1981, 68(5): 1321-1330.
24. Cohick WA, Clemmons DR. The insulin-like growth factors[J]. Annu Rev Physiol, 1993, 55:131-153.
25. 李丽艳, 路新国. 胰岛素样生长因子系统与运动系统的关系研究进展[J]. 中国康复医学杂志, 2005, 20(8):632-635.
26. 陈照丽,王欣,黄秉人. IGF 系统的生物学功能及其与肿瘤的关系 [J]. 国外医学分子生物学分册,2003,25(1):34-37.
27. 刘宝英,王会信. 胰岛素样生长因子 1 的结构与功能研究进展[J]. 生物化学与生物物理进展,1998,25(4):311-315.
28.Akihiro S, Shigenori N, Tadayasu O et al. Three-dimensional structure of human insulin-like growth factor-1(IGF1)detemined by 1H-NMR and distance geometry[J]. Int J Peptide Protein Res, 1993, 41(5):433-440.
29. Bayne ML, Applebaum J, Chicchi CG et al. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and type 2 insulin-like growth factor receptor[J]. J Biol Chem, 1988, 263(13):6233-6239.
30. Cascieri MA, Chicchi GC, Applebaum J et al. Structural analogs of human insulin-like growth factor(IGF) 1 with altered affinity for type 2 IGF receptor[J]. J Biol Chem, 1989, 264(4):2199-2202.
31. Morgan DO, Edman JC, Standring DN et al. Insulin-like growth factor 2 receptor as a multifunctional bindig protein[J]. Nature, 1987, 329(24):301-307.
32. Rajaram S, Baylink DJ, Mohan S. Insulin-like growth factor binding proteins in serum and other biological fluids: regulation and functions[J]. Endocr Rev, 1997, 18:801-831.
33. 高玉红,李庆章. 胰岛素生长因子系统研究进展[J]. 中国乳品工业,2006, 34(4):46-49.
34. Victor KMH, Douglas GM, Patric JDD et al. IGF-binding protein mRNAs in the human fetus, Tissue and cellular distribution of developmental expression[J]. Human Res, 1996, 45(1):160-166.
35. Clemmons DR, Moses AC, Mckay MJ,etal. The combination of insulin-like growth factor 1 and insulin-like growth factor bindingprotein-3 reduces insulin requirements in insulin-dependent type 1 diabetes: evidence for invivo biological activity[J]. J Clin Endocrinol Metab, 2000, 85:1518-1524.
36. 段宇,刘超,汪承亚,等. 胰岛素样生长因子 1 研究进展[J]. 国外医学内分泌学分册,2001,21(6):305-307.
37. Rosen CJ, Conover C. GH/IGF-1 axis in aging: a summary of a NIA-sponsored symposium[J]. J Clin Endocrinol Metab, 1997, 82:3919-3922.
38. Estivariz CF, Zieyler TR. Nutrition and the insulin-like growth factor system. Endocrine, 1997, 7:65-71.
39. Dunger DB, Cheetham TD. Growth hormone insulin-like growth factor I axis in insulin-dependent diabetes mellitus. Horm Res, 1996, 46:2-6.
40. Harrela M, Kao PC, Matheny AP, et al. Genetic and environmentalcomponents of inter-individual variation in circulating levels of IGF-1, IGF-2, IGFBP-1, IGFBP-3[J]. J Clin Invest, 1996, 98:2612-2615.
41. Rosen CJ, Conover C. Circulating and skeletal IGF-1 concentrations in two inbred strains of mice with different bone densities[J]. Bone, 1997, 21:217-223.
42. Rosen CJ, Kurland ES, Vereauit D, et al. Association between serum IGF-1 and a simple sequence repeat in the IGF-1 gene: implications for genetic studies of BMD[J]. J Clin Endocrinol Metab, 1998, 83:228622290.
43. Neuberg M, Buckbinder L, Seizinger B, et al. The P53/IGF-1 receptor axis in the regulation of programmed cell death[J]. Endocrine, 1997, 138:1072109.
44. 杜惠峰, 傅中滇. 糖尿病及其国内流行现状[J]. 生物学教,2002,27(1):4-5.
45. 王克安. 中国糖尿病的防治和研究[J]. 中华流行病学杂志,1999,20(5):260-263.
46. Alan DC. 糖尿病:过去、现在和将来[J]. 国外医学内分泌学分册,2005, 25(5):附录 3-5.
47. 韩炳鑫,李晓东,刘小虎,等. 第 65 届美国糖尿病年会纪要[J]. 中国全科医学,2006,9(16):1385-1386.
48. Cusz K, Defronzo RA. Treatment of NIDDM, IDDM and other insulin-resistent states with IGF-1[J]. Diabetes Rev, 1998, 3:206-229.
49. Dunger DB, Cheetham TD. Holly JMP. Does recombinant insulin-like growth factor I have a role in the treatment of IDDM during adolescence[J]. Acta Paediatr, 1993, 338(Suppl): 49-52.
50. 许辉,蒙碧辉. IGF-1 在Ⅰ型糖尿病中的研究进展[J]. 医学研究杂志,2006,35(12):79-81.
51. Clauson PG, Brismar K, Hall K, et al. Insulin-like growth factor-I andinsulin-like growth factor binding protein-1 in a representative population of type 2 diabetic patients in Sweden [J], Scand J Clin Lab Invest, 1998, 58:353-360.
52. 张金梅,张洁,刘金玲,等. 胰岛素样生长因子 1 与糖尿病及其并发症的关系[J], 中西医结合心脑血管病杂志,2007,5(1):58-59.
53. Sasaoka T, Kobayashi M. Takata Y, et al. Clarification of signaling pathways mediated by insulin and insulin-like growth factor I receptors in fibroblasts from patients with specific defect in insulin receptor [J]. Diabetes, 1998, 37:1515-1523.
54. Kato M. Biological and clinical analyses of the mechanism of growth retardation and the effect of recombinant human insulin-like growth factor-1(rhIGF-1) treatment on glucose metabolism and growth in leprechaunism with severe insulin resistance diabetes [J]. Hokkaido Igaku Zasshi, 1998, 73:613-625.
55. Laron Z, Anin S, Klipper-Aurbach Y, et al. Effects of insulin-like growth factors on linear growth, head circumference, and body fat in patients with Laron-type dwarfism[J]. Lancet, 1992, 339:1258-1261.
56. Brain CE, Hubbard M, Preece MA, et al. Metabolic states of children with growth hormone insensitivity syndrome and responses to treatment with HHIGF-1 [J]. Horm Res, 1998, 50:61-70.
57. Jehle P, Schulten K, Schulz W, et al. Serum levels of insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 to -6 and their relationship to bone metabolism in osteoporosis patients. Eur J Intern Med, 2003, 14:32238.
58. Reinecke M, Schmid AC, Hunziker EB, et al. Effect of growth hormone and insulin-like growth factor I (IGF-1) on the expression of IGF-1 messenger ribonucleic acid and peptide in rat tibial growth plate and articular chondrocytes in vivo. Endocrinology, 2000, 141:2847-2853.
59. Miyatake N Shikata K, Wada J, et al. Differential distribution of insulin-like growth factor-1 and insulin-like growth factor binding proteins in experimental diabetic rat kidney [J]. Nephron, 1999, 81:317-323.
60. Gambaro G, Baggio B. Growth factors and the kidney in diabetes mellitus[J]. Crit Rev Clin Lab Sci. 1998, 35:117-151.
61. Herndon DN, Ramzy PI, Debroy MA, et al. Muscle protein catabolism after severe burn: effects of IGF-1/IGFBP-3 treatment[J]. Ann Surg. 1999, 229:713-722.
62. Mauras N, O’Brien KO, Welch S, et al. Insulin-like growth factor I and growth hormone(GH) treatment in GH-deficient humans: differential effects on protein, glucose, lipid, and calcium metabolism[J].J Clin Endocrinol Metab 2000, 85:1686-1694.
63. Ishii DN. Implication of insulin-like growth factors in the pathogenesis of diabetic neuropathy[J]. Brain Res Revi, 1995, 20:47267.
64. Torresaleman I, Barrios V, Berciano J. The peripheral insulin-like growth factor system in amyotrophic lateral sclerosis and in multiple sclerosis[J]. Neurology, 1998, 50: 772-776.
65. 徐伟文,吴岚晓, 李明. 胰岛素样生长因子-1 的临床应用及其前景[J]. 国外医学内分泌学分册,1999,19(6):270-273.
66. Kovacs GT. Worgall S, Schwalbach P, et al. Hypoglycemic effects of insulin-like growth factor-1 in experimental uremia: can concomitant growth hormone administration prevent this effect [J]. Horm Res, 1999,51:193-200.
67. Roith DL. Insulin-like growth factors[J]. N Engl J Med, 1997, 336: 633-640.
68. Acerini CL, Patton CM, Savage MO, et al. Randomised placebo-controlled trial of human recombinant insulin-like growth factor-1 plus intensive insulin therapy in adolescents with insulin-dependent diabetes mellitus[J]. Lancet, 1997, 350:1199-1204.
69. Kuroki M, Kawakami MD. Diabetic retinopathy-the mechanisms of the ocular neovascularization and the development of antiangiogenic drugs [J]: Nippon Rinsho, 1999. 57:584-589.
70. http://www.d1.com.cn/html/01101231.htm
71. 黄胜炎. 国外新批准上市的药物新制剂新剂型[J]. 中国制药信息,2006,22(4):23-26.
72. www.mofcom.gov.cn/aarticle/o/di/200603/20060301743202.html
73. 陆志城. 医药经济报,2006 年医院周刊第 21-23 期.
74. Lewis ME, Neff NT, Contreras PC et al. Insulin-like growth faetor I: Potential for treatment of motor neuronal disorders[J]. Exptl Neurol, 1993, 124:73-88.
75. 聂尚海、刘宝英. 胰岛素样生长因子 I 与肌萎缩性侧索硬化症[J].神经生化学通讯,1998,11(3,4):77-81.
76. Fervenza. Insulin-like growth factor-I treatment to enhance renal function in advanced chronic renal failure [J], Ren Fail,1998,20:349-354.
77. Yarwood. Administration of human recombinant insulin-like growth factor-I in critically ill patients [J].Crit Care Med, 1997, 25:1352-1356.
78. 李伯良、胡明、杨新颖等. 人胰岛素生长因子基因人工合成、克隆及其表达[J]. 生物化学与生物物理学报, 1994,26(2):153-160.
79. 李军、李伯良. 蛋白 A 信号肽引导的 E.coli 外分泌高表达异源蛋白[J].生物化学与生物物理学报,1995,27(6)616-623.
80. 刘宝英、赵明、王会信等. 人胰岛素样生长因子 I 在大肠杆菌中的表达研究[J]. 中国生物化学与分子生物学报, 1998,14(1):47-51.
81、刘宝英、聂尚海、杜清友等. 人胰岛素样生长因子 I 突变体的构建、表达及其鉴定[J]. 军事医学科学院院刊,1999,23(2):94-96.
82. 梁东春,左爱军,吴亚锋,等. 重组人胰岛素样生长因子 I 的制备[J]. 天津医药, 2005,33(7):434-436.
83. 凌明圣、许祥裕、施凤霞等. 人胰岛素样生长因子 I 基因的半合成及克隆表达[J]. 中国生化药物杂志, 1999,20(4):166-169.
84. 吴岚晓,李 明,王 萍,等. 人胰岛素样生长因子-1 在大肠杆菌中的表达[J]. 药物生物技术,2001,8(4):185-188.
85. 吴岚晓,李明,陈白虹,等. 重组人胰岛素样生长因子-1 分离纯化工艺的建立[J]. 中国药学杂志,2002,37(5):376-379.
86. 高媛,余榕捷,洪岸,等. 人类胰岛素生长因子基因的合成、表达与纯化[J]. 中国病理生理杂志,2005,21(2):260-265.
87. 刘侃,汪炬,谢秋玲,等. 重组类胰岛素样生长因子-1 的纯化与复性 [J]. 中国生物工程杂志, 2006,26(2):29-33.
88. 魏辉俊、黄秉仁、蔡良琬. 类胰岛素生长因子 I 的克隆、表达、分离纯化与活性检测[J]. 中国医学科学院学报, 1997,19(5):342-345.
89. 赖心田,周鹏,洪葵. 人胰岛素样生长因子-1 在毕赤酵母中的表达研究 [J].药物生物技术,2002,9(3):133-136.
90. 刘晓辉、梁述文、王伟. 截短型人胰岛素样生长因子 rIGF-1 在大肠杆菌中的表达,纯化和鉴定[J]. 中国生物化学与分子生物学报,1999,15(5):738-741.
91. 李莹、邓鸿业、狄春辉等. 重组人截短型胰岛素样生长因子 I 在大肠杆菌中的高效表达[J]. 中国免疫学杂志, 2000,16(2):57-60.
92. 周鹏,王跃进,贺普超,等. 人胰岛素样生长因子-1 基因农杆菌工程菌株的构建[J]. 西北农林科技大学学报,2001,29(3):19-23.
93. 张英华,王小佳. 用于转化甘蓝的胰岛素样生长因子 I 的合成及MAR 调控的表达载体构建[J]. 农业生物技术学报,2004,12(6):731-732.
94. 赵红、 汪承亚、陈华群,等. 昆虫细胞表达重组人胰岛素样生长因子 I 的研究[J]. 南京医科大学学报,2000,20(4):243-245.
95. 段 宇,汪承亚,赵 红,等. 利用杆状病毒载体在家蚕幼虫中高效表达人胰岛素样生长因子-1 的研究[J]. 中国生化药物杂志,2001,22(6):274-277.
96. 徐志伟,娄亚欣,李 莹,等. 人胰岛素样生长因子 1 的真核细胞表达及其鉴定[J]. 免疫学杂志,2001,17(1):1-3.
97. 岳凤鸣,赵焕英,杨慧,等. 人胰岛素样生长因子-1 基因的克隆、表达和活性检测[J]. 解剖学报,2003,34(3):306-311.
98. 张绍昆,刘 一,单玉兴,等. 人胰岛素样生长因子-1 稳定表达细胞株的构建及鉴定[J].吉林大学学报 (医学版) ,2003,29(1):41-43.
99. 张海宁,侯筱魁,杨冠珍,等. 人 IGF-1 基因真核表达质粒pIRES2-EGFP- IGF-1 的构建[J]. 上海第二医科大学学报,2004,24(5):359-361.
100. 祁雅慧,王雅梅,孙丽翠,等. 人胰岛素样生长因子-1 基因的克隆、表达及生物活性分析[J]. 首都医科大学学报,2004,25(2):154-158.
101. 张鹏,陈世益,陈疾忤,等. 人胰岛素样生长因子-1 真核表达质粒的构建及其在成肌细胞中的表达 [J].中国运动医学杂志,2005,24(1):9-20.
102. 周海斌,郑祖根,董启榕. 人胰岛素样生长因子-1 的真核表达及其生物学功能研究[J].中国病理生理杂志,2007,23(1):27-29.