PEG修饰干扰素的合成与生物活性的研究
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摘要
目的:为了改善干扰素(IFN)的稳定性和生物学活性,采用高分子设计原理,以活化的聚乙二醇(PEG)来修饰干扰素,以几种不同的PEG修饰剂对干扰素分别进行修饰,比较不同PEG及不同修饰率修饰干扰素与未修饰干扰素在热稳定性、酶稳定性、耐酸碱性方面的差异,并对产物进行抗病毒性能与抑制肿瘤细胞增殖性能的检测。
方法:以顺丁烯二酸酐(MAn)和烯丙基化合物为单体,经自由基共聚制备了大分子量的PEG为长侧链的聚合物来修饰干扰素。采用SDS-PAGE电泳法测定PEG修饰干扰素的分子量,以证实合成产物是否与理论设计相符;通过考察热稳定性、酶稳定性、耐酸碱性来比较PEG修饰干扰素与未修饰干扰素之间性能的差异;用Hep-2细胞-VSV病毒体系检测合成产物抗病毒性能;采用MTT法测定合成产物对肿瘤细胞增殖的影响。
结果:以不同的修饰剂和不同的修饰率制备了九种PEG修饰IFN;DS-PAGE电泳结果表明有聚合物-IFN结合物生成,分子量为6万(以修饰剂APEO90—4万修饰)的产物三种、分子量为4万(以修饰剂APEO90—2万与APEO60—2万修饰)的产物六种; PEG修饰干扰素的热稳定性、酶稳定性、耐酸碱性比未修饰干扰素均有明显的增加;MTT法实验表明PEG修饰的IFN对肿瘤细胞生长抑制作用比未修饰IFN增强,9种PEG修饰IFN(产物编号1-9)分别提高了56.3%、35.2%、17.8%、42.3%、23.5%、14.6%、45.5%、26.5%、11.4%,而其抗病毒性能则有不同程度的下降,依次下降了52%、78%、88%、48%、78%、89%、54%、79%、88%。
结论:PEG修饰干扰素的热稳定性、酶稳定性、耐酸碱性均有显著的提高;PEG修饰干扰素的抗病毒性能下降;PEG修饰干扰素的抗肿瘤细胞增殖作用优于未修饰干扰素。
OBJECTIVE: To improve the stability and biological activity, interferon can be modified by activated Polyethylene glycol (PEG) with the macromolecular design. To synthesize PEGylated IFN with a series of PEG modifier, compare their differences of heat stability, enzyme stability and stability of acid base. It was detected to antiviral activities and antineoplastic activities of PEGylated IFN.
METHODS: The comblike copolymers of polycarboxylic acid (CPC) were synthesized and then reacted with PEG, obtained a conjugate-- PEGylated IFN. It was determined the molecular weight of conjugate by SDS-PAGE. The difference of heat stability, enzyme stability, and stability of acid base were compared between the PEGylated IFN and IFN. Antiviral activities can be detected by the system of Hep-2 cell and VSV. MTT assay was applied to assess antineoplastic activities of PEGylated IFN.
RESULTS: There were 9 kinds of PEG-IFN conjugates, which were modified by various modifier and different modifying. The results of SDS-PAGE showed that the PEG-IFN conjugates were synthesized. The molecular weight of conjugate was 60000(modified by APEO90-4000) and 40000(modified by APEO90-20000 and APEO60-2000). The heat stability, enzyme stability and stability of acid base were increased conspicuously. MTT assay showed that PEG-IFN can inhibit the proliferation of tumor cells. Inhibitory effect of the 9 kinds of PEG-IFN (marked with 1-9) was increased. The increased rates were 56.3%, 35.2%, 17.8%, 42.3%, 23.5%, 14.6%,45.5%, 26.5%, 11.4% respectively. But antiviral activity of PEG-IFN was decreased. The decreased rates were 52%, 78%, 88%, 48%, 78%, 89%, 54%, 79%, 88% respectively.
CONCLUSION: The heat stability, enzyme stability and stability of acid base of products were increased conspicuously. The antiviral effect of PEGylated IFN was not more effective than which of IFN. The effect of PEG-IFN was better than the effect of IFN in inhibiting tumor cells.
方法:以顺丁烯二酸酐(MAn)和烯丙基化合物为单体,经自由基共聚制备了大分子量的PEG为长侧链的聚合物来修饰干扰素。采用SDS-PAGE电泳法测定PEG修饰干扰素的分子量,以证实合成产物是否与理论设计相符;通过考察热稳定性、酶稳定性、耐酸碱性来比较PEG修饰干扰素与未修饰干扰素之间性能的差异;用Hep-2细胞-VSV病毒体系检测合成产物抗病毒性能;采用MTT法测定合成产物对肿瘤细胞增殖的影响。
结果:以不同的修饰剂和不同的修饰率制备了九种PEG修饰IFN;DS-PAGE电泳结果表明有聚合物-IFN结合物生成,分子量为6万(以修饰剂APEO90—4万修饰)的产物三种、分子量为4万(以修饰剂APEO90—2万与APEO60—2万修饰)的产物六种; PEG修饰干扰素的热稳定性、酶稳定性、耐酸碱性比未修饰干扰素均有明显的增加;MTT法实验表明PEG修饰的IFN对肿瘤细胞生长抑制作用比未修饰IFN增强,9种PEG修饰IFN(产物编号1-9)分别提高了56.3%、35.2%、17.8%、42.3%、23.5%、14.6%、45.5%、26.5%、11.4%,而其抗病毒性能则有不同程度的下降,依次下降了52%、78%、88%、48%、78%、89%、54%、79%、88%。
结论:PEG修饰干扰素的热稳定性、酶稳定性、耐酸碱性均有显著的提高;PEG修饰干扰素的抗病毒性能下降;PEG修饰干扰素的抗肿瘤细胞增殖作用优于未修饰干扰素。
OBJECTIVE: To improve the stability and biological activity, interferon can be modified by activated Polyethylene glycol (PEG) with the macromolecular design. To synthesize PEGylated IFN with a series of PEG modifier, compare their differences of heat stability, enzyme stability and stability of acid base. It was detected to antiviral activities and antineoplastic activities of PEGylated IFN.
METHODS: The comblike copolymers of polycarboxylic acid (CPC) were synthesized and then reacted with PEG, obtained a conjugate-- PEGylated IFN. It was determined the molecular weight of conjugate by SDS-PAGE. The difference of heat stability, enzyme stability, and stability of acid base were compared between the PEGylated IFN and IFN. Antiviral activities can be detected by the system of Hep-2 cell and VSV. MTT assay was applied to assess antineoplastic activities of PEGylated IFN.
RESULTS: There were 9 kinds of PEG-IFN conjugates, which were modified by various modifier and different modifying. The results of SDS-PAGE showed that the PEG-IFN conjugates were synthesized. The molecular weight of conjugate was 60000(modified by APEO90-4000) and 40000(modified by APEO90-20000 and APEO60-2000). The heat stability, enzyme stability and stability of acid base were increased conspicuously. MTT assay showed that PEG-IFN can inhibit the proliferation of tumor cells. Inhibitory effect of the 9 kinds of PEG-IFN (marked with 1-9) was increased. The increased rates were 56.3%, 35.2%, 17.8%, 42.3%, 23.5%, 14.6%,45.5%, 26.5%, 11.4% respectively. But antiviral activity of PEG-IFN was decreased. The decreased rates were 52%, 78%, 88%, 48%, 78%, 89%, 54%, 79%, 88% respectively.
CONCLUSION: The heat stability, enzyme stability and stability of acid base of products were increased conspicuously. The antiviral effect of PEGylated IFN was not more effective than which of IFN. The effect of PEG-IFN was better than the effect of IFN in inhibiting tumor cells.
引文
1、Feeney R E.Chemical modification of proteins: comment and perspectives. Int. J. Peptide Protein Res., 1987, 29(2):145-161.
2、Haney C R, Burbler P W, Gulati A. Purification and chemical modifications of hemoglobin in developing hemoglobin based oxygen carriers. Adv.Drug Deliv. Rev, 2000, 40(3):153-169.
3、Tam JW ,Cheng L Y .Chemical cross-linking of hemoglobin H ,a possible approach to introduce cooperativity and modification of its oxygen transport properties. Biochim. Biophys.Acta, 1979, 580(1):75-84.
4、印春华,张敏.蛋白质和多肽类药物的聚乙二醇结合物——一种新型给药系统.中国药学杂志,2001,36:292-296.
5、Kozlowski A, Harris JM. Improvements in protein PEGylation: pegylation interferon for treatment of hepatitis C. J Control Release, 2001,72: 217-224.
6、姜忠义,高蓉,王艳强,等.蛋白质和多肽药物聚乙二醇化的问题与对策.药学学报,2002.37(5):396-400.
7、周海梦,王洪睿.蛋白质化学修饰.北京:清华大学出版社,1998.102-108.
8、Clark R, Olson K, Flu G, et al. Long-acting growth hormones produced by conjugation with polyethylene glycol. Biol Chem, 1996, 271: 21969 -21977.
9、Delgado C, Francis GE, Fisher D. The use and properties of PEG-Linked proteins. The Drug Carries Syst, 1992, 9:249-304.
10、公瑞煜,李建蓉,石朝周等.聚羧酸型梳状共聚物超分散剂的构性关系.化工学报,2002,53(11):1143-1147.
11、公瑞煜,李建蓉,张化锋,等.聚羧酸型梳状共聚物的合成和超分散性能研究.功能材料,2004, 35(3):379-382.
12、公瑞煜,李建蓉,王洛礼等.水溶性梳状聚合物超分散剂的结构与其对水泥浆分散性能关系的研究.精细石油化工,2003,(1):50-54.
13、张龙翔,张庭芳,李令媛.生化实验方法和技术.北京:高等教育出版社,1997.137-138.
14、Mosmann T .Rapid calorimetric assay for cellular g rowth and surviv al: application to proliferation and cytotoxicity assays.J Immunal Methods,1983,05,55-63.
15、司徒镇强,吴军正.细胞培养.西安:世界图书出版社,1996,40-43.
16、付春晓.蛋白的聚乙二醇修饰及其在生物医学领域的应用.国外医学药学分册,2001,28 (2):79-80.
17、Herold D A, Keil K and Bruns D E. Oxidation of polyethylene glycols by alcohol dehydrogenase. Biochem.Pharmacol,1989,38: 73-76.
18、Lee V HL. Peptide and protein drug delivery: opportunities and challenges. Pharm In,1986,8: 208-211.
19、Jenog ST, Byun SM, Decreased agglutinability of methoxy-polyethylene glycol attached red blood cells: significance as a blood substitute. Artif Cells Blood Submit Immobil Biotechnol, 1996, 24: 503-511.
20、B.D.哈密斯,D.利克伍德.蛋白质的凝胶电泳.北京:科学出版社,1994,151-157.
21、杜平.医用干扰素学.北京:解放军出版社,1985,221-228.
22、侯云德,吴淑华.干扰素.北京:人民卫生出版社,1981,67-71.
23、侯云德.干扰素及其临床应用.南京:江苏科学技术出版社,1981,102-105.
24、姚文兵,林碧蓉,沈子龙等.聚乙二醇修饰干扰素α2b的体内药代动力学研究.中国药科大学学报.2001,32(6):457-461.
25、盛新华,卿三华,李镏洋.MTT、W ST 28法测定细胞生长抑制率的结果比较.山东医药2006 ,46(6):20-21.
26、房云,杨锦蓉,王菁.MTT法观察DMF致人肝细胞增殖抑制作用.浙江预防医学2006,18(11):1-2.
27、安飞云,廖春华,边寰峰.改良MTT法检测HePG22细胞增殖的研究.毒理学杂志.2006,20(3):186-187.
28、方蓉,李芳秋,武建国.MTT比色法的条件探讨.临床检验杂志2003,21(1):34-35.
29、黄志锋,郑青,苏志坚等.聚乙二醇定点修饰haFGF突变体.化学通报2004(67):1-5.
30、刘长暖,刘兰,王军志,郭莹,丁锡申.应用结晶紫染色法测定干扰素效价.中国生物制品学杂志,1999,12(1):36-38.
31、徐静,倪道明,张振龙.聚乙二醇修饰β-干扰素的研究.微生物学免疫学进展,2004,32(3):30-33.
32、刘丽军,赵丽纯,朱迅,石晓丽,刘丹,张岩,张雪梅,盛军,聚乙二醇活性酯对干扰素α2b化学修饰的初步研究.中国生物制品学杂志,2001,14(4):210-212.
33、Francesca J. Torriani, M.D., Maribel Rodriguez-Torres, M.D., Jurgen K.Rockstroh, M.D, et al.Peginterferon Alfa-2a plus Ribavirin for Chronic Hepatitis C Virus Infection in HIV-Infected Patients.The New England Journal of Medicine,2004,5(351):438-450.
34、Carrat, F., Bani-Sadr, F., Pol, S., Rosenthal, E. et al.Pegylated Interferon Alfa-2b vs Standard Interferon Alfa-2b, Plus Ribavirin, for Chronic Hepatitis C in HIV-Infected Patients: A Randomized Controlled Trial. JAMA, 2004, 292:2839-2848.
35、Taylor,L. E., Rich, J. D., Tashima, K. T. et al. Pegylated Interferon Alfa-2a in Patients with HCV and HIV. Journal Watch Gastroenterology,2004:5-5.
36、J Shepherd, H Brodin, C Cave, N Waugh et al. Pegylated interferon alpha-2a and -2b in combination with ribavirin in the treatment of chronic hepatitis C: a systematic review and economic evaluation. Health Technology Assessment,2004 8(39):111-125.
37、Kontorinis N, Agarwal K, Elhajj N et al. Pegylated interferon-induced immune-mediated hepatitis post-liver transplantation. Liver Transpl. 2006,12(5):827-830.
38、von Wagner M, Huber M, Berg T, Peginterferon-alpha-2a (40KD) and ribavirin for 16 or 24 weeks in patients with genotype 2 or 3 chronic hepatitis C. Gastroenterology. 2005,129(2):522-527.
39、Xie L, Cullen C, Bradshaw S et al. Biological characterization of pegylated interferons: a case study. Methods Mol Biol. 2005,308:319-335.
40、Eric Snoeck,Janet R. Wade, Frank Duff et al. Predicting sustained virological response and anaemia in chronic hepatitis C patients treated with peginterferon alfa-2a (40KD) plus ribavirin. British Journal of Clinical Pharmacology,2006,62(6):699-709.
41、黄志荣.MTT比色法在干扰素生物学活性测定中应用的探讨.海峡药学,2006,18(6):60-61.
42、张明娟,方娟,胡太发.聚乙二醇α-2a干扰素与α-1b干扰素治疗慢性乙型肝炎疗效比较.宁夏医学院学报,2006,28(6):532-533.
1、付春晓.蛋白的聚乙二醇修饰及其在生物医学领域的应用.国外医学药学分册.2001,28 (2):79-80.
2、Kozlowski A, Harris JM. Improvements in protein PEGylation: pegylation interferons for treatment of hepatitisC. J Control Release, 2001,72: 217-224.
3、姜忠义,高蓉,王艳强,等.蛋白质和多肽药物聚乙二醇化的问题与对策.药学学报,2002,37(5):396-400.
4、周海梦,王洪睿.蛋白质化学修饰.北京:清华大学出版社,1998,1.
5、Harrs J M ,Zalipsky S. Poly(ethylene Glycol Chemistry and Biological Applications. Washington: American Chemical Society,1997,1-474.
6、Lundblad R L ,Bradshaw R A .Application of site-specitic chemical modification in the manufacture of biopharmaceuticals:Ⅰ.An overview. Biotechnol.Appl.Biochem,1997,26(3):143-151.
7、Inada Y, Furukawa M, Sasaki H, Kodera Y, Hiroto M, Nishimura A. Biomedical and biotechnological applications of PEG-and PM-modified proteins. Trends Biotechnol,1995,13(3):86-91.
8、Abuchowski A, Van E T, et al. Alteration of immunological properties of bovine serum albumin by covalent attachment of poly(ethylene glycol)J Biol Chem,1977,252:3578-3586
9、Rameshraja P, Hua Shan W. Current Aspects in pharmawlogy of Modified Hemoglobins. Adv Drug Deliv Review,2000,40:185-198.
10、付春晓.蛋白的聚乙二醇修饰及其在生物医学领域的应用.国外医学药学分册.2001,28(2):79-80.
11、Habbeb ASFA. Determination of free amino groups in proteins by trinitrobenzene-sulfonic acid. Anal Biochem,1996,14:328-336
12、Guermant C, Brygier B B, Looze Y. Quantiative determination of polyethylene glycol based upon its salting out and partitioning of a dye into the resulting aqueous two phase system. Anal Biochem,1995,230:254- 260.
13、Jackson C J, Kuzminski K. Synthesis isolation and characterization of conjugations of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the couping agent.Anal Biochem,1987,165(1):114-127.
14、Kurfurst M.M. Detection and Molecular Weight Determination of Polyethylene Glyco-Modified Hirudin by Staining after Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.Anal Biochem,1992,200:244- 248.
15、McGoff P., BaziotisA.C. and Maskiewicz R.Analysis of Polyethylene Glycol Modified Superoxide Dismutase by Chromatographic,Electrophoretic, Light Scattering, Chemical and Enzymatic Methods. Chem.Pharm.Bull,1988, 36:3079-3091.
16、M J Robert s ,M D Bentley ,J M Harris. Advanced Drug Delivery Reviews,2002 ,54 :459-476
2、Haney C R, Burbler P W, Gulati A. Purification and chemical modifications of hemoglobin in developing hemoglobin based oxygen carriers. Adv.Drug Deliv. Rev, 2000, 40(3):153-169.
3、Tam JW ,Cheng L Y .Chemical cross-linking of hemoglobin H ,a possible approach to introduce cooperativity and modification of its oxygen transport properties. Biochim. Biophys.Acta, 1979, 580(1):75-84.
4、印春华,张敏.蛋白质和多肽类药物的聚乙二醇结合物——一种新型给药系统.中国药学杂志,2001,36:292-296.
5、Kozlowski A, Harris JM. Improvements in protein PEGylation: pegylation interferon for treatment of hepatitis C. J Control Release, 2001,72: 217-224.
6、姜忠义,高蓉,王艳强,等.蛋白质和多肽药物聚乙二醇化的问题与对策.药学学报,2002.37(5):396-400.
7、周海梦,王洪睿.蛋白质化学修饰.北京:清华大学出版社,1998.102-108.
8、Clark R, Olson K, Flu G, et al. Long-acting growth hormones produced by conjugation with polyethylene glycol. Biol Chem, 1996, 271: 21969 -21977.
9、Delgado C, Francis GE, Fisher D. The use and properties of PEG-Linked proteins. The Drug Carries Syst, 1992, 9:249-304.
10、公瑞煜,李建蓉,石朝周等.聚羧酸型梳状共聚物超分散剂的构性关系.化工学报,2002,53(11):1143-1147.
11、公瑞煜,李建蓉,张化锋,等.聚羧酸型梳状共聚物的合成和超分散性能研究.功能材料,2004, 35(3):379-382.
12、公瑞煜,李建蓉,王洛礼等.水溶性梳状聚合物超分散剂的结构与其对水泥浆分散性能关系的研究.精细石油化工,2003,(1):50-54.
13、张龙翔,张庭芳,李令媛.生化实验方法和技术.北京:高等教育出版社,1997.137-138.
14、Mosmann T .Rapid calorimetric assay for cellular g rowth and surviv al: application to proliferation and cytotoxicity assays.J Immunal Methods,1983,05,55-63.
15、司徒镇强,吴军正.细胞培养.西安:世界图书出版社,1996,40-43.
16、付春晓.蛋白的聚乙二醇修饰及其在生物医学领域的应用.国外医学药学分册,2001,28 (2):79-80.
17、Herold D A, Keil K and Bruns D E. Oxidation of polyethylene glycols by alcohol dehydrogenase. Biochem.Pharmacol,1989,38: 73-76.
18、Lee V HL. Peptide and protein drug delivery: opportunities and challenges. Pharm In,1986,8: 208-211.
19、Jenog ST, Byun SM, Decreased agglutinability of methoxy-polyethylene glycol attached red blood cells: significance as a blood substitute. Artif Cells Blood Submit Immobil Biotechnol, 1996, 24: 503-511.
20、B.D.哈密斯,D.利克伍德.蛋白质的凝胶电泳.北京:科学出版社,1994,151-157.
21、杜平.医用干扰素学.北京:解放军出版社,1985,221-228.
22、侯云德,吴淑华.干扰素.北京:人民卫生出版社,1981,67-71.
23、侯云德.干扰素及其临床应用.南京:江苏科学技术出版社,1981,102-105.
24、姚文兵,林碧蓉,沈子龙等.聚乙二醇修饰干扰素α2b的体内药代动力学研究.中国药科大学学报.2001,32(6):457-461.
25、盛新华,卿三华,李镏洋.MTT、W ST 28法测定细胞生长抑制率的结果比较.山东医药2006 ,46(6):20-21.
26、房云,杨锦蓉,王菁.MTT法观察DMF致人肝细胞增殖抑制作用.浙江预防医学2006,18(11):1-2.
27、安飞云,廖春华,边寰峰.改良MTT法检测HePG22细胞增殖的研究.毒理学杂志.2006,20(3):186-187.
28、方蓉,李芳秋,武建国.MTT比色法的条件探讨.临床检验杂志2003,21(1):34-35.
29、黄志锋,郑青,苏志坚等.聚乙二醇定点修饰haFGF突变体.化学通报2004(67):1-5.
30、刘长暖,刘兰,王军志,郭莹,丁锡申.应用结晶紫染色法测定干扰素效价.中国生物制品学杂志,1999,12(1):36-38.
31、徐静,倪道明,张振龙.聚乙二醇修饰β-干扰素的研究.微生物学免疫学进展,2004,32(3):30-33.
32、刘丽军,赵丽纯,朱迅,石晓丽,刘丹,张岩,张雪梅,盛军,聚乙二醇活性酯对干扰素α2b化学修饰的初步研究.中国生物制品学杂志,2001,14(4):210-212.
33、Francesca J. Torriani, M.D., Maribel Rodriguez-Torres, M.D., Jurgen K.Rockstroh, M.D, et al.Peginterferon Alfa-2a plus Ribavirin for Chronic Hepatitis C Virus Infection in HIV-Infected Patients.The New England Journal of Medicine,2004,5(351):438-450.
34、Carrat, F., Bani-Sadr, F., Pol, S., Rosenthal, E. et al.Pegylated Interferon Alfa-2b vs Standard Interferon Alfa-2b, Plus Ribavirin, for Chronic Hepatitis C in HIV-Infected Patients: A Randomized Controlled Trial. JAMA, 2004, 292:2839-2848.
35、Taylor,L. E., Rich, J. D., Tashima, K. T. et al. Pegylated Interferon Alfa-2a in Patients with HCV and HIV. Journal Watch Gastroenterology,2004:5-5.
36、J Shepherd, H Brodin, C Cave, N Waugh et al. Pegylated interferon alpha-2a and -2b in combination with ribavirin in the treatment of chronic hepatitis C: a systematic review and economic evaluation. Health Technology Assessment,2004 8(39):111-125.
37、Kontorinis N, Agarwal K, Elhajj N et al. Pegylated interferon-induced immune-mediated hepatitis post-liver transplantation. Liver Transpl. 2006,12(5):827-830.
38、von Wagner M, Huber M, Berg T, Peginterferon-alpha-2a (40KD) and ribavirin for 16 or 24 weeks in patients with genotype 2 or 3 chronic hepatitis C. Gastroenterology. 2005,129(2):522-527.
39、Xie L, Cullen C, Bradshaw S et al. Biological characterization of pegylated interferons: a case study. Methods Mol Biol. 2005,308:319-335.
40、Eric Snoeck,Janet R. Wade, Frank Duff et al. Predicting sustained virological response and anaemia in chronic hepatitis C patients treated with peginterferon alfa-2a (40KD) plus ribavirin. British Journal of Clinical Pharmacology,2006,62(6):699-709.
41、黄志荣.MTT比色法在干扰素生物学活性测定中应用的探讨.海峡药学,2006,18(6):60-61.
42、张明娟,方娟,胡太发.聚乙二醇α-2a干扰素与α-1b干扰素治疗慢性乙型肝炎疗效比较.宁夏医学院学报,2006,28(6):532-533.
1、付春晓.蛋白的聚乙二醇修饰及其在生物医学领域的应用.国外医学药学分册.2001,28 (2):79-80.
2、Kozlowski A, Harris JM. Improvements in protein PEGylation: pegylation interferons for treatment of hepatitisC. J Control Release, 2001,72: 217-224.
3、姜忠义,高蓉,王艳强,等.蛋白质和多肽药物聚乙二醇化的问题与对策.药学学报,2002,37(5):396-400.
4、周海梦,王洪睿.蛋白质化学修饰.北京:清华大学出版社,1998,1.
5、Harrs J M ,Zalipsky S. Poly(ethylene Glycol Chemistry and Biological Applications. Washington: American Chemical Society,1997,1-474.
6、Lundblad R L ,Bradshaw R A .Application of site-specitic chemical modification in the manufacture of biopharmaceuticals:Ⅰ.An overview. Biotechnol.Appl.Biochem,1997,26(3):143-151.
7、Inada Y, Furukawa M, Sasaki H, Kodera Y, Hiroto M, Nishimura A. Biomedical and biotechnological applications of PEG-and PM-modified proteins. Trends Biotechnol,1995,13(3):86-91.
8、Abuchowski A, Van E T, et al. Alteration of immunological properties of bovine serum albumin by covalent attachment of poly(ethylene glycol)J Biol Chem,1977,252:3578-3586
9、Rameshraja P, Hua Shan W. Current Aspects in pharmawlogy of Modified Hemoglobins. Adv Drug Deliv Review,2000,40:185-198.
10、付春晓.蛋白的聚乙二醇修饰及其在生物医学领域的应用.国外医学药学分册.2001,28(2):79-80.
11、Habbeb ASFA. Determination of free amino groups in proteins by trinitrobenzene-sulfonic acid. Anal Biochem,1996,14:328-336
12、Guermant C, Brygier B B, Looze Y. Quantiative determination of polyethylene glycol based upon its salting out and partitioning of a dye into the resulting aqueous two phase system. Anal Biochem,1995,230:254- 260.
13、Jackson C J, Kuzminski K. Synthesis isolation and characterization of conjugations of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the couping agent.Anal Biochem,1987,165(1):114-127.
14、Kurfurst M.M. Detection and Molecular Weight Determination of Polyethylene Glyco-Modified Hirudin by Staining after Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.Anal Biochem,1992,200:244- 248.
15、McGoff P., BaziotisA.C. and Maskiewicz R.Analysis of Polyethylene Glycol Modified Superoxide Dismutase by Chromatographic,Electrophoretic, Light Scattering, Chemical and Enzymatic Methods. Chem.Pharm.Bull,1988, 36:3079-3091.
16、M J Robert s ,M D Bentley ,J M Harris. Advanced Drug Delivery Reviews,2002 ,54 :459-476