人巨噬细胞金属弹力酶催化域基因克隆、表达、纯化与活性鉴定
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摘要
目的本研究通过构建人巨噬细胞金属弹力酶催化域(HMEcd)基因原核表达质粒,诱导大肠杆菌表达融合6×His tag的HMEcd基因融合蛋白,经纯化、复性后用于HME体内外干预胃癌细胞实验研究。
方法提取手术切除的新鲜胃癌组织总RNA,逆转录合成cDNA第一链,设计一对特异性引物PCR扩增HMEcd cDNA,1%琼脂糖凝胶电泳鉴定并切胶纯化后与pMD18-T载体连接,构建克隆质粒pMD18-T-HMEcd,转化大肠杆菌DH5α,提取质粒,BamH I和Xho I双酶切鉴定及DNA测序正确后再将HMEcd cDNA亚克隆至pET-28a(+)载体,构建原核表达质粒pET-28a(+)-HMEcd,转化大肠杆菌DH5α,提取质粒,BamH I和Xho I双酶切鉴定及DNA测序后,转化大肠杆菌BL21(DE3)诱导表达,SDS-PAGE及Western blot鉴定。His Band镍柱亲和纯化,透析重折叠复性,浓缩后测定蛋白质浓度,明胶酶谱分析及I型胶原蛋白分解实验鉴定酶活性。
结果RT-PCR获得预期的HMEcd cDNA,双酶切鉴定及DNA测序证实分别成功构建克隆质粒pMD18-T-HMEcd和原核表达质粒pET-28a(+)-HMEcd,SDS-PAGE及Western blot证实在大肠杆菌中诱导表达出HMEcd基因融合蛋白,诱导5h蛋白表达量占菌体总蛋白的18.92%。纯化后SDS-PAGE分析蛋白质纯度达98%以上。重折叠复性后经活性鉴定证实HMEcd基因融合蛋白具有分解明胶及I型胶原蛋白的酶活性。
结论获得HMEcd cDNA,成功构建原核表达质粒;在大肠杆菌中高效表达出HMEcd基因融合蛋白;建立融合蛋白亲和纯化及体外重折叠复性方法,成功获得高纯度活性HMEcd基因融合蛋白;为下一步HME对胃癌细胞的体内外干预研究创造了条件。
Objective In this experiment we cloned 507 bp cDNA fragment coding for human macrophage metalloelastase catalytic domain (HMEcd) and constructed a prokaryotic expression plasmid and expressed it in E. coli.Then HMEcd protein was purified and renatured, which can be used in the studies on tumor intervention with HME.
Methods HMEcd cDNA was amplified by reverse transcriptional polymerase chain reaction(RT-PCR) from tissue of gastric cancer and identified by 1% agarose gel electrophoresis. The fragment was inserted into pMD18-T vector and constructed pMD18-T-HMEcd plasmid. The plasmid was transformed into E. coli DH5αand amplified in the bacterium. Then we extracted pMD18-T-HMEcd plasmid from E. coli DH5αand identified the recombinant plasmid by double digestion with restriction enzymes BamH I and Xho I and sequencing. HMEcd cDNA cutted from pMD18-T-HMEcd was subcloned into pET-28a(+) vector. The recombinant pET-28a(+)-HMEcd was identified with the same method for pMD18-T-HMEcd and transformed into E. coli BL21(DE3) and induced by IPTG. The production of expression was analyzed by SDS-PAGE and detected by Western-blot. The fusion protein was purified with His Bind Collumn and refolded by dialysis and assaied after concentration. The enzymatic activity of recombinant protein was confirmed by gelatin zymography and cleavage of type I collagen in vitro.
Results HMEcd cDNA was cloned. The pMD18-T-HMEcd and pET-28a(+)-HMEcd plasmid were conducted and confirmed. SDS-PAGE and Western-blot demonstrated HMEcd protein was expressed in E. coli BL-21(DE3). The recombinant protein occupied approximately 18.92% of total bacterial protein. HMEcd protein was purified with a purity of over 98% and demonstrated the enzymatic activity.
Conclusions We obtained HMEcd cDNA and prokaryotic expressive plasmid perfectly.The recombinant HMEcd fusion protein was expressed successfully. We established the purification and refolding scheme through the experiments. Which laid the foundation for successive studies.
方法提取手术切除的新鲜胃癌组织总RNA,逆转录合成cDNA第一链,设计一对特异性引物PCR扩增HMEcd cDNA,1%琼脂糖凝胶电泳鉴定并切胶纯化后与pMD18-T载体连接,构建克隆质粒pMD18-T-HMEcd,转化大肠杆菌DH5α,提取质粒,BamH I和Xho I双酶切鉴定及DNA测序正确后再将HMEcd cDNA亚克隆至pET-28a(+)载体,构建原核表达质粒pET-28a(+)-HMEcd,转化大肠杆菌DH5α,提取质粒,BamH I和Xho I双酶切鉴定及DNA测序后,转化大肠杆菌BL21(DE3)诱导表达,SDS-PAGE及Western blot鉴定。His Band镍柱亲和纯化,透析重折叠复性,浓缩后测定蛋白质浓度,明胶酶谱分析及I型胶原蛋白分解实验鉴定酶活性。
结果RT-PCR获得预期的HMEcd cDNA,双酶切鉴定及DNA测序证实分别成功构建克隆质粒pMD18-T-HMEcd和原核表达质粒pET-28a(+)-HMEcd,SDS-PAGE及Western blot证实在大肠杆菌中诱导表达出HMEcd基因融合蛋白,诱导5h蛋白表达量占菌体总蛋白的18.92%。纯化后SDS-PAGE分析蛋白质纯度达98%以上。重折叠复性后经活性鉴定证实HMEcd基因融合蛋白具有分解明胶及I型胶原蛋白的酶活性。
结论获得HMEcd cDNA,成功构建原核表达质粒;在大肠杆菌中高效表达出HMEcd基因融合蛋白;建立融合蛋白亲和纯化及体外重折叠复性方法,成功获得高纯度活性HMEcd基因融合蛋白;为下一步HME对胃癌细胞的体内外干预研究创造了条件。
Objective In this experiment we cloned 507 bp cDNA fragment coding for human macrophage metalloelastase catalytic domain (HMEcd) and constructed a prokaryotic expression plasmid and expressed it in E. coli.Then HMEcd protein was purified and renatured, which can be used in the studies on tumor intervention with HME.
Methods HMEcd cDNA was amplified by reverse transcriptional polymerase chain reaction(RT-PCR) from tissue of gastric cancer and identified by 1% agarose gel electrophoresis. The fragment was inserted into pMD18-T vector and constructed pMD18-T-HMEcd plasmid. The plasmid was transformed into E. coli DH5αand amplified in the bacterium. Then we extracted pMD18-T-HMEcd plasmid from E. coli DH5αand identified the recombinant plasmid by double digestion with restriction enzymes BamH I and Xho I and sequencing. HMEcd cDNA cutted from pMD18-T-HMEcd was subcloned into pET-28a(+) vector. The recombinant pET-28a(+)-HMEcd was identified with the same method for pMD18-T-HMEcd and transformed into E. coli BL21(DE3) and induced by IPTG. The production of expression was analyzed by SDS-PAGE and detected by Western-blot. The fusion protein was purified with His Bind Collumn and refolded by dialysis and assaied after concentration. The enzymatic activity of recombinant protein was confirmed by gelatin zymography and cleavage of type I collagen in vitro.
Results HMEcd cDNA was cloned. The pMD18-T-HMEcd and pET-28a(+)-HMEcd plasmid were conducted and confirmed. SDS-PAGE and Western-blot demonstrated HMEcd protein was expressed in E. coli BL-21(DE3). The recombinant protein occupied approximately 18.92% of total bacterial protein. HMEcd protein was purified with a purity of over 98% and demonstrated the enzymatic activity.
Conclusions We obtained HMEcd cDNA and prokaryotic expressive plasmid perfectly.The recombinant HMEcd fusion protein was expressed successfully. We established the purification and refolding scheme through the experiments. Which laid the foundation for successive studies.
引文
1. Folkman J.Tumor angiogenesis:therapeutic implications. N Engl J Med. 1971, 285: 1182-1186.
2. Folkman J.Anti-angiogenesis:New concept of therapy of solid tumors.Ann Surg, 1972,175:409-416.
3. Gastl G ,Hermann T ,Steurer M ,et al .Angiogenesis as a target for tumor treatment.Oncogen,1977,54:177.
4. Sato H, Takino T, Okada Y,et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells.Nature,1994,370(6484):61-5.
5. 张金库.基质金属蛋白酶及其抑制剂与肿瘤关系的研究进展.诊断病理学杂志, 2004,11(4):269-271.
6. Gorrin-Rivas MJ, Arii S, Mori A, et al. Implications of human macrophage metalloelastase and vascular endothelial growth factor gene expression in angiogenesis of hepatocellular carcinoma. Ann Surg, 2000,231(1):67-73.
7. Shapiro SD, Griffin GL, Gilbert DJ, et al. Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase. J Biol Chem, 1992, 267(14): 4664-4671.
8. Shapiro SD,Kobayashi DK,Ley TJ. Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophage. J Biol Chem, 1993,268(32): 23824-9.
9. Dong Z,Kumar R,Yang X,et al. Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell,1997,88(6): 801-810.
10. Cornelius LA,Nehring LC,Harding E.Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immunol.1998,161(12): 6845-52.
11. Gorrin-Rivas MJ, Arii S, Furutani M,et al. Expression of human macrophage metalloelastase gene in hepatocellular carcinoma: correlation with angiostatin generation and its clinical significance. Hepatology,1998; 28(4):986-993.
12. Salmela MT, Karjalainen-Lindsberg ML, Puolakkainen P, et al.Upregulation and differential expression of matrilysin (MMP-7) and metalloelastase (MMP-12) and their inhibitors TIMP-1 and TIMP-3 in Barrett's oesophageal adenocarcinoma. Br J Cancer,2001,85(3):383-92.
13. Yang W, Arii S, Gorrin-Rivas MJ et al. Human macrophage metalloelastase gene expression in colorectal carcinoma and its clinicopathologic significance.Cancer, 2001,91(7):1277-1283.
14. Shi H,Xu J M,Hu N Z,et al. Transfection of mouse macrophage metalloelastase gene into murine CT-26 colon cancer cells suppresses orthotopic tumor growth, angiogenesis and vascular endothelial growth factor expression.Cancer Lett,2006, 233(1):139-50.
15. Normann SJ. Macrophage infiltration and tumor progression. Cancer and Metas Rev, 1985,4(1): 277-291.
16. Vukanovic J and Isaacs JT. Linomide inhibits angiogenesis, growth, metastasis, and macrophage infiltration within rat prostatic cancers.Cancer Res,1995,55(6): 1499-1504.
17. Kachra Z,Beaulieu E,Delbecchi L,et al. Expression of matrix metalloproteinases and their inhibitors in human brain tumors.Clin Exp Metastasis.1999,17(7): 555-66.
18. Balaz P, Friess H, Kondo Y,et al. Human macrophage metalloelastase worsens theprognosis of pancreatic cancer.Ann Surg, 2002,235(4):519-527.
19. Liu W, Xiang C, Jia S.Study on the expression of matrix metalloproteinase-12 and NM_002426 in squamous laryngeal carcinoma.Lin Chuang Er Bi Yan Hou Ke Za Zhi.2006,20(24):1120-3.
20. Parkar AA, Stow MD, Smith K,et al. Large-scale expression, refolding, and purification of the catalytic domain of human macrophage metalloelastase (MMP-12) in Escherichia coli. Protein Expr Purif. 2000,20(2):152-61.
21. Hefti MH, Van Vugt-Van der Toorn CJ, Dixon R,et al. A novel purification method for histidine-tagged proteins containing a thrombin cleavage site. Anal Biochem. 2001,295(2):180-5.
22. Lilie H, Schwarz E, Rudolph R. Advances in refolding of proteins produced in E. coli. Curr Opin Biotechnol. 1998,9(5):497-501.
23. 白东亭,许丽峰.重组大肠杆菌的包涵体.中国生物制品学杂,1996,9(4):188-190.
24. Li M, Su ZG, Janson JC. In vitro protein refolding by chromatographic procedures. Protein Expr Purif. 2004,33(1):1-10.
25. XieY ,Wetlaufer DB. Control of aggregation in protein refolding: the temperature-leap tactic.Protein Sci,1996,5(3):517-23.
26. Fischer B,Sumner 1,Goodenough P. Isolation, renaturation, and formation of disulfide bonds of eakarytic proteins expressed in Escherichia coli as inclusion bodies.Biotechnology and Bioengineering,1993,41(1):3-13.
27. D.R.马歇克,J.T.门永,R.R.布格斯等著,朱厚础等译.蛋白质纯化与鉴定实验指南.科学出版社,2000,18.
28. 吴二喜,王凤飞,Norman McKIE.基质金属蛋白酶.生命科学研究,1999,3(3): 175-194.
29. 孙红霞,韩梅,温进坤.用含底物SDS-PAGE检测明胶酶活性.基础医学与临床.2000,20(5):94-96.
30. Kleiner DE, Stetler-Stevenson WG.Quantitative zymography:detection of pictogram quantities of gelatinases. Anal Biochem.1994,218(2):325-9.
1. Folkman J.Tumor angiogenesis:therapeutic implications.N Engl J Med,1971, 285 (21): 1182-6.
2. Folkman J.Anti-angiogenesis:New concept of therapy of solid tumors.Ann Surg,1972,175(3):409-16.
3. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metallo-proteinases:structure, function, and biochemistry.Circ Res, 2003,92(8): 827-39.
4. 张金库.基质金属蛋白酶及其抑制剂与肿瘤关系的研究进展.诊断病理学杂志,2004,11(4):269-271.
5. Shapiro SD, Griffin GL, Gilbert DJ et al. Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase. J Biol Chem,1992,267(14): 4664-4671
6. Shapiro SD, Kobayashi DK, Ley TJ. Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophage. J Biol Chem, 1993,268(32):23824-9.
7. Chandler S,Cossins J,Lury J,et al.Macrophage metalloelastase degrades matrix and myelin proteins and processes a tumour necrosis factor-alpha fusion protein. Biochem Biophys Res Commun,1996,228(2):421-9.
8. Gronski TJ Jr,Martin RL,Kobayashi DK,et al. Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase. J Biol Chem,1997, 272(18):12189-94.
9. Kumar R,Dong Z,Fidler IJ.Differential regulation of metalloelastase activity in murine peritoneal macrophages by granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor.J Immunol,1996,157(11): 5104-11.
10. Horton MR,Shapiro S,Bao C,et al.Induction and regulation of macrophage metalloelastase by hyaluronan fragments in mouse macrophages.J Immunol,1999, 162(7):4171-6.
11. Belaaouaj A,Shipley JM,Kobayashi DK,et al. Human macrophage metalloelastase. Genomic organization,chromosomal location,gene linkage and tissue-specific expression.J Biol Chem,1995,270(24):14568-75.
12. Feinberg MW,Jain MK,Werner F,et al.Transforming growth factor-beta 1 inhibits cytokine-mediated induction of human metalloelastase in macrophages.J Biol Chem, 2000,275(33):25766-73.
13. Kerkela E,Bohling T,Herva R,et al. Human macrophage metalloelastase (MMP-12) expression is induced in chondrocytes during fetal development and malignant transformation. Bone,2001,29(5):487-93
14. Raza SL,Nehring LC,Shapiro SD,et al. Proteinase-activated receptor-1 regulation of macrophage elastase(MMP-12) secretion by serine proteinases.J Biol Chem, 2000,275(52):41243-50.
15. O'Reilly MS,Holmgren L,Shing Y,et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma.Cell,1994, 79(2):315-28.
16. Cornelius LA,Nehring LC,Harding E,et al. Matrix metalloproteinases generate angiostatin: effects on neovascularization.J Immunol,1998,161(12):6845-52.
17. Dong Z,Kumar R,Yang X,et al. Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma.Cell,1997,88(6): 801-10.
18. Gorrin-Rivas MJ,Arii S,Furutani M,et al. Expression of human macrophage metalloelastase gene in hepatocellular carcinoma: correlation with angiostatin generation and its clinical significance.Hepatology,1998,28(4):986-93.
19. Kim S,Park HS,Son HJ,et al.The role of angiostatin, vascular endothelial growth factor,matrix metalloproteinase 9 and 12 in the angiogenesis of hepatocellular carcinoma.Korean J Hepatol,2004,10(1):62-72.
20. Ferreras M,Felbor U,Lenhard T,et al. Generation and degradation of human endostatin proteins by various proteinases.FEBS Lett,2000,486(3):247-51.
21. O'Reily MS,Boehm T,Shing Y,et al. Endostatin:an endogenous inhibitor of angio-genesis and tumor growth.Cell,1997,88(2):277-85.
22. Gorrin-Rivas MJ,Arii S,Furutani M,et al. Mouse macrophage metalloelastase gene transfer into a murine melanoma suppresses primary tumor growth by halting angiogenesis.Clin Cancer Res,2000,6(5):1647-54.
23. Kerkela E,Ala-AhoR,Jeskanen L,et al. Expression of human macrophage metallo-elastase (MMP-12) by tumor cells in skin cancer.J Invest Dermatol,2000,114(6):1113-1119.
24. Salmela MT,Karjalainen-Lindsberg ML,Puolakkainen P,et al.Upregulation and differential expression of matrilysin (MMP-7) and metalloelastase (MMP-12) and their inhibitors TIMP-1 and TIMP-3 in Barrett's oesophageal adenocarcinoma.Br J Cancer,2001,85(3):383-92.
25. Gorrin-Rivas M J,Arii S,Mori A,et al. Implications of human macrophage metallo- elastase and vascular endothelial growth factor gene expression in angiogenesis of hepatocellular carcinoma.Ann Surg,2000,231(1):67-73.
26. Yang W,Arii S,Gorrin-Rivas M J,et al. Human macrophage metalloelastase gene expression in colorectal carcinoma and its clinicopathologic significance.Cancer,2001,91(7):1277-83.
27. Shi H,Xu J M,Hu N Z,et al. Transfection of mouse macrophage metalloelastase gene into murine CT-26 colon cancer cells suppresses orthotopic tumor growth, angiogenesis and vascular endothelial growth factor expression.Cancer Lett,2006, 233(1): 139-50.
28. Kachra Z,Beaulieu E,Delbecchi L,et al. Expression of matrix metalloproteinases and their inhibitors in human brain tumors.Clin Exp Metastasis.1999,17(7):555-66.
29. Balaz P,Friess H,Kondo Y,et al. Human macrophage metalloelastase worsens the prognosis of pancreatic cancer.Ann Surg, 2002,235(4):519-27
30. Liu W,Xiang C,Jia S.Study on the expression of matrix metalloproteinase-12 and NM_002426 in squamous laryngeal carcinoma.Lin Chuang Er Bi Yan Hou Ke Za Zhi.2006,20(24):1120-3.
2. Folkman J.Anti-angiogenesis:New concept of therapy of solid tumors.Ann Surg, 1972,175:409-416.
3. Gastl G ,Hermann T ,Steurer M ,et al .Angiogenesis as a target for tumor treatment.Oncogen,1977,54:177.
4. Sato H, Takino T, Okada Y,et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells.Nature,1994,370(6484):61-5.
5. 张金库.基质金属蛋白酶及其抑制剂与肿瘤关系的研究进展.诊断病理学杂志, 2004,11(4):269-271.
6. Gorrin-Rivas MJ, Arii S, Mori A, et al. Implications of human macrophage metalloelastase and vascular endothelial growth factor gene expression in angiogenesis of hepatocellular carcinoma. Ann Surg, 2000,231(1):67-73.
7. Shapiro SD, Griffin GL, Gilbert DJ, et al. Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase. J Biol Chem, 1992, 267(14): 4664-4671.
8. Shapiro SD,Kobayashi DK,Ley TJ. Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophage. J Biol Chem, 1993,268(32): 23824-9.
9. Dong Z,Kumar R,Yang X,et al. Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell,1997,88(6): 801-810.
10. Cornelius LA,Nehring LC,Harding E.Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immunol.1998,161(12): 6845-52.
11. Gorrin-Rivas MJ, Arii S, Furutani M,et al. Expression of human macrophage metalloelastase gene in hepatocellular carcinoma: correlation with angiostatin generation and its clinical significance. Hepatology,1998; 28(4):986-993.
12. Salmela MT, Karjalainen-Lindsberg ML, Puolakkainen P, et al.Upregulation and differential expression of matrilysin (MMP-7) and metalloelastase (MMP-12) and their inhibitors TIMP-1 and TIMP-3 in Barrett's oesophageal adenocarcinoma. Br J Cancer,2001,85(3):383-92.
13. Yang W, Arii S, Gorrin-Rivas MJ et al. Human macrophage metalloelastase gene expression in colorectal carcinoma and its clinicopathologic significance.Cancer, 2001,91(7):1277-1283.
14. Shi H,Xu J M,Hu N Z,et al. Transfection of mouse macrophage metalloelastase gene into murine CT-26 colon cancer cells suppresses orthotopic tumor growth, angiogenesis and vascular endothelial growth factor expression.Cancer Lett,2006, 233(1):139-50.
15. Normann SJ. Macrophage infiltration and tumor progression. Cancer and Metas Rev, 1985,4(1): 277-291.
16. Vukanovic J and Isaacs JT. Linomide inhibits angiogenesis, growth, metastasis, and macrophage infiltration within rat prostatic cancers.Cancer Res,1995,55(6): 1499-1504.
17. Kachra Z,Beaulieu E,Delbecchi L,et al. Expression of matrix metalloproteinases and their inhibitors in human brain tumors.Clin Exp Metastasis.1999,17(7): 555-66.
18. Balaz P, Friess H, Kondo Y,et al. Human macrophage metalloelastase worsens theprognosis of pancreatic cancer.Ann Surg, 2002,235(4):519-527.
19. Liu W, Xiang C, Jia S.Study on the expression of matrix metalloproteinase-12 and NM_002426 in squamous laryngeal carcinoma.Lin Chuang Er Bi Yan Hou Ke Za Zhi.2006,20(24):1120-3.
20. Parkar AA, Stow MD, Smith K,et al. Large-scale expression, refolding, and purification of the catalytic domain of human macrophage metalloelastase (MMP-12) in Escherichia coli. Protein Expr Purif. 2000,20(2):152-61.
21. Hefti MH, Van Vugt-Van der Toorn CJ, Dixon R,et al. A novel purification method for histidine-tagged proteins containing a thrombin cleavage site. Anal Biochem. 2001,295(2):180-5.
22. Lilie H, Schwarz E, Rudolph R. Advances in refolding of proteins produced in E. coli. Curr Opin Biotechnol. 1998,9(5):497-501.
23. 白东亭,许丽峰.重组大肠杆菌的包涵体.中国生物制品学杂,1996,9(4):188-190.
24. Li M, Su ZG, Janson JC. In vitro protein refolding by chromatographic procedures. Protein Expr Purif. 2004,33(1):1-10.
25. XieY ,Wetlaufer DB. Control of aggregation in protein refolding: the temperature-leap tactic.Protein Sci,1996,5(3):517-23.
26. Fischer B,Sumner 1,Goodenough P. Isolation, renaturation, and formation of disulfide bonds of eakarytic proteins expressed in Escherichia coli as inclusion bodies.Biotechnology and Bioengineering,1993,41(1):3-13.
27. D.R.马歇克,J.T.门永,R.R.布格斯等著,朱厚础等译.蛋白质纯化与鉴定实验指南.科学出版社,2000,18.
28. 吴二喜,王凤飞,Norman McKIE.基质金属蛋白酶.生命科学研究,1999,3(3): 175-194.
29. 孙红霞,韩梅,温进坤.用含底物SDS-PAGE检测明胶酶活性.基础医学与临床.2000,20(5):94-96.
30. Kleiner DE, Stetler-Stevenson WG.Quantitative zymography:detection of pictogram quantities of gelatinases. Anal Biochem.1994,218(2):325-9.
1. Folkman J.Tumor angiogenesis:therapeutic implications.N Engl J Med,1971, 285 (21): 1182-6.
2. Folkman J.Anti-angiogenesis:New concept of therapy of solid tumors.Ann Surg,1972,175(3):409-16.
3. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metallo-proteinases:structure, function, and biochemistry.Circ Res, 2003,92(8): 827-39.
4. 张金库.基质金属蛋白酶及其抑制剂与肿瘤关系的研究进展.诊断病理学杂志,2004,11(4):269-271.
5. Shapiro SD, Griffin GL, Gilbert DJ et al. Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase. J Biol Chem,1992,267(14): 4664-4671
6. Shapiro SD, Kobayashi DK, Ley TJ. Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophage. J Biol Chem, 1993,268(32):23824-9.
7. Chandler S,Cossins J,Lury J,et al.Macrophage metalloelastase degrades matrix and myelin proteins and processes a tumour necrosis factor-alpha fusion protein. Biochem Biophys Res Commun,1996,228(2):421-9.
8. Gronski TJ Jr,Martin RL,Kobayashi DK,et al. Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase. J Biol Chem,1997, 272(18):12189-94.
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