从中华眼镜蛇蛇毒中分离纯化神经生长因子的色谱工艺研究
|
摘要
本研究论文以中华眼镜蛇蛇毒中神经生长因子的分离纯化为目的，以广东产中华眼镜蛇粗毒为开发对象，用不同的色谱介质联用的方法进行工艺研究，寻找一种可以简单、快速、分离效果好且可几乎线性放大的纯化工艺。论文的研究工作为中华眼镜蛇蛇毒中NGF的工业化生产以及蛇毒的综合利用开辟了新的途径。
论文在实验、研究过程中，采用了6种不同的色谱介质进行组合，共设计了5种不同的分离方案对中华眼镜蛇蛇毒NGF进行纯化，实验发现采用DEAE-Sepharose Fast Flow阴离子交换层析与Sephadex G50凝胶过滤层析联用的两步色谱法可以从蛇毒中得到纯度可达药用标准的目标产品。此工艺较传统方法具有分离速度快、工艺简单、可线性放大的特点。色谱条件优化实验确定阴离子的色谱条件为：洗脱流速0.5mL／min，洗脱液A为0.05mol/L PBS，pH8.5，洗脱液B为0.25mol/LNaCl+0.05mol/L PBS，pH8.5，洗脱程序为等度洗脱+线性梯度洗脱+等度洗脱的淋洗方式。凝胶过滤层析的色谱条件为：洗脱流速0.9mL／min，洗脱液A为0.05mol/L PBS，pH7.5，洗脱程序为等度洗脱。纯化后含NGF的蛋白溶液经Brodford法测定蛋白质的浓度为16ng／mL，回收率为0.49％；以凝胶过滤和反相两种高效液相色谱法测定其纯度，纯度都在98％以上，可以达到药用标准；采用SDS-PAGE电泳法也证明所得的蛋白溶液只有一条蛋白带，其分子质量约为14000；鸡胚背根神经节体外培养法证明，纯化所得蛋白溶液在浓度为9ng／mL时即可维持神经纤维的最大生长。对分离得到的NGF蛋白溶液进行小白鼠急性毒性试验和长期毒性实验证明，中华眼镜蛇蛇毒中纯化的NGF蛋白急性毒性和长期毒性在小白鼠体内为阴性。
采用两步色谱法分离纯化蛇毒神经生长因子所得的目标产品经分析，各项指标不低于传统方法所报道的水平，部分指标优于文献报道。
Nerve growth factor (NGF) is a protein necessary for the maintenance and development of sympathetic and sensory neurons. It can not only promote the fission of neuron but also determine the growth direction of neurons. It also plays an important physiological role in central nervous system. So therapy with NGF for neurological disorders, such as Alzheimer's disease, Parkinson's disease, etc., is valuable.
To identify a simple, fast and high efficient method for separation and purification of nerve growth factor (NGF) from venom of Chinese Cobra, the combination process of several different kinds of chromatographic media was studied. According to the properties of NGF and the character of different chromatographic media, a novel two-step chromatographic purification method, consisting of chromatography of crude venom on DEAE-Sepharose F.F. anion-exchange medium followed by a size exclusion on Sephadex G-50, is presented in this paper. The DEAE-Sepharose F.F. chromatographic column was 20cm×3.5cm i.d. and first eluted with the 50mmol/L Tris-HCl (pH 8.5) for 50 min, and separately followed by the elution with a linear gradient of 0-200mmol/L NaCl containing 50mmol/L Tris-HCl (pH 8.5) for 190min and that with 200mmol/L NaCl containing 50mmol/L Tris-HCl (pH 8.5) for 60min. The flow-rate of mobile phase was 6.1ml/min. The Sephadex G 50 column was 150 cm×3.5cm i.d. and eluted with 50mmol/L PBS (pH 7.5) for 1250min. The flow-rate of elution solution was 2.4ml/min. Through this two-step chromatographic purification process, the obtained NGF was proved to be homogeneous on SDS-PAGE and its molecular weight was estimated to be approximately 14000, which was consistent with that reported in literatures; and on Shim-pack VP-ODS reversed-phase high-performance chromatographic column(4.6 mm i.d.×150mm), its purity was about 97.5%. The total protein recovery of this purification method is 0.51% and the obtained NGF had the activity of eliciting neurite outgrowth from chick embryonic dorsal root ganglia. This two-step chromatographic process is simple and high efficient, and the indexes of aim prodction are no less than the traditional method's, it can be used to isolate and purify NGF from venom of Chinese Cobra in large-scale.
Peng Wu (Biochemical Engineering) Directed by Bian Liujiao
论文在实验、研究过程中，采用了6种不同的色谱介质进行组合，共设计了5种不同的分离方案对中华眼镜蛇蛇毒NGF进行纯化，实验发现采用DEAE-Sepharose Fast Flow阴离子交换层析与Sephadex G50凝胶过滤层析联用的两步色谱法可以从蛇毒中得到纯度可达药用标准的目标产品。此工艺较传统方法具有分离速度快、工艺简单、可线性放大的特点。色谱条件优化实验确定阴离子的色谱条件为：洗脱流速0.5mL／min，洗脱液A为0.05mol/L PBS，pH8.5，洗脱液B为0.25mol/LNaCl+0.05mol/L PBS，pH8.5，洗脱程序为等度洗脱+线性梯度洗脱+等度洗脱的淋洗方式。凝胶过滤层析的色谱条件为：洗脱流速0.9mL／min，洗脱液A为0.05mol/L PBS，pH7.5，洗脱程序为等度洗脱。纯化后含NGF的蛋白溶液经Brodford法测定蛋白质的浓度为16ng／mL，回收率为0.49％；以凝胶过滤和反相两种高效液相色谱法测定其纯度，纯度都在98％以上，可以达到药用标准；采用SDS-PAGE电泳法也证明所得的蛋白溶液只有一条蛋白带，其分子质量约为14000；鸡胚背根神经节体外培养法证明，纯化所得蛋白溶液在浓度为9ng／mL时即可维持神经纤维的最大生长。对分离得到的NGF蛋白溶液进行小白鼠急性毒性试验和长期毒性实验证明，中华眼镜蛇蛇毒中纯化的NGF蛋白急性毒性和长期毒性在小白鼠体内为阴性。
采用两步色谱法分离纯化蛇毒神经生长因子所得的目标产品经分析，各项指标不低于传统方法所报道的水平，部分指标优于文献报道。
Nerve growth factor (NGF) is a protein necessary for the maintenance and development of sympathetic and sensory neurons. It can not only promote the fission of neuron but also determine the growth direction of neurons. It also plays an important physiological role in central nervous system. So therapy with NGF for neurological disorders, such as Alzheimer's disease, Parkinson's disease, etc., is valuable.
To identify a simple, fast and high efficient method for separation and purification of nerve growth factor (NGF) from venom of Chinese Cobra, the combination process of several different kinds of chromatographic media was studied. According to the properties of NGF and the character of different chromatographic media, a novel two-step chromatographic purification method, consisting of chromatography of crude venom on DEAE-Sepharose F.F. anion-exchange medium followed by a size exclusion on Sephadex G-50, is presented in this paper. The DEAE-Sepharose F.F. chromatographic column was 20cm×3.5cm i.d. and first eluted with the 50mmol/L Tris-HCl (pH 8.5) for 50 min, and separately followed by the elution with a linear gradient of 0-200mmol/L NaCl containing 50mmol/L Tris-HCl (pH 8.5) for 190min and that with 200mmol/L NaCl containing 50mmol/L Tris-HCl (pH 8.5) for 60min. The flow-rate of mobile phase was 6.1ml/min. The Sephadex G 50 column was 150 cm×3.5cm i.d. and eluted with 50mmol/L PBS (pH 7.5) for 1250min. The flow-rate of elution solution was 2.4ml/min. Through this two-step chromatographic purification process, the obtained NGF was proved to be homogeneous on SDS-PAGE and its molecular weight was estimated to be approximately 14000, which was consistent with that reported in literatures; and on Shim-pack VP-ODS reversed-phase high-performance chromatographic column(4.6 mm i.d.×150mm), its purity was about 97.5%. The total protein recovery of this purification method is 0.51% and the obtained NGF had the activity of eliciting neurite outgrowth from chick embryonic dorsal root ganglia. This two-step chromatographic process is simple and high efficient, and the indexes of aim prodction are no less than the traditional method's, it can be used to isolate and purify NGF from venom of Chinese Cobra in large-scale.
Peng Wu (Biochemical Engineering) Directed by Bian Liujiao
引文
[1]Bucker E.D., Anat. Rec., 1948, 102:369-390
[2]Levi-Montalcini R. et.al, 1951,166:321-362
[3]Cohen S. et.al, Proc. Natl. Acad. Sci. U.S.A., 1954,40:1014-1018
[4]Cohen S. Levi-Montalcini R., Proc. Natl. Acad. Sci. U.S.A., 1956,42:571-574
[5]Levi-Montalcini R., Cohen S., Ibid., 1956,42:695-699
[6]Cohen S.,Symposium Chem.Basis Develop.,Johns Hopkins Univ., 1958:665-676
[7]Cohen S., J. Biol. Chem.,1959, 234:1129-1137
[8]Cohen S., Proc. Natl. Acad. Sci., U.S.A.,1967, 57:1782-1789
[9]Thoenen H., Barde Y.A., Physiol. Rev., 1980, 60:1284
[10]Senut M.C, Lamour Y., J. Lee, P. Brachet, F. Dicou, Neurosecience, 1990, 8:65
[11]Goedert M., Finc A., dawbarn D., Wilcock G.K., Mol. Brain Res., 1989, 5:1
[12]Gibbs R.B., McCabe J.T., Buck C. R, Chao M.V., Pfaff D.W., Mol. Brain Res.,1989, 6:275
[13]Moble W.C., Rutkowski J.L., Jennckoon G.I., Buckanank, Science, 1985, 229:284
[14]Helti F., Neurosci J., 1986, 6:2155
[15]Mark T., James C., Armin B., David S., Pro. Brain Res., 2002, 138:401
[16]Simona C., Sabina G., Antonino C., Mol. Cellular Neurosci., 2002, 21:15
[17]Hellweg H., Hartung H.D., J. Neurosci. Res. 1990, 26:258
[18]李素芹、李法卿、谭维国等，中国生化药物杂志．1999，20：123
[19]祁岩超、周旻、李志阳等，广东医学．1996，17：694
[20]Nishizawa M, Ozawa F, Higashizakit T, Hirai K, Hishinuma F, Appl Microbiol Biotechnol, 1993, 88:624-630
[21]Dicon E, Neurochem Int, 1992, 20:129-134
[22]Lwane M, Biochemi Biophysi Res Commu, 1990, 171:116-122
[23]郭立英，刘杰武，周元聪等，生物化学与生物物理学报，1999，31(2)：203-206
[24]Hogue-Angeletti R.A., J. chromatogr., 1968, 36:535
[25]Hogue-Angeletti R.A., J. chromatogr., 1968, 37:62
[26]Hogue-Angeletti R.A., Frazier W.A., Jacobs, H. J.W. Niall D., Bradshaw R.A., Biochemistry, 1976, 15:26-34
[27]Furukawsa S., Hayashi K., J. Biochem., 1976, 80:1001
[28]Khamidov D.K., Salikhov R.S., Khafizova M.G. et al, Khim. Prir. Soedin., 1985, 4:546
[29]Siigur J., Arumae V., Neuman T. et al, Comp. Biochem. Physiol.,B: Comp.Biochem., 1986, 83B: 621
[30]SiigurG.et.al,Comp.Biochem.Physiol.,B :Comp.Biochem, 1987,87B: 329-334
[31]D. K. Khamidov, L.Y. Ywkelson, R. S.Salikhov et al, Biokhimiya, 1989, 54:987
[32]雷少波，吴秀荣，生物化学与生物物理学报，1989，21：115-119
[33]Smith RJ., Brandt W.F., Stickells B.J. et al, Comp. Biochem. Phusiol., B: Comp. Biochem., 1992, 103B: 975
[34]Kilnion J., Am. Biotechnol. Lab., 1992, 10:18
[35]潘清，郝文学，发明专利申请公开说明书，CN 1，074，448，1992(9)
[36]Koyama J.I., Inoue S., Ikeda K. et al, Biochim. Biophys. Acta, 1992, 1160:787
[37]Horie S., Inoue S., Ikeda K. et al, J. Nat. Toxins, 1994, 3:165
[38]Kostiza T., Dahinden C.A., Rihs S. et al, Toxico., 1995, 33:1249
[39]刘一平，李虹，王锡峰，中国生化药物杂志，1994，15：111-113
[40]刘一平，潘华，李虹等，中国生化药物杂志，1996，17：238-241
[41]刘春宇，陈洁，卢军等，暨南大学学报(自然科学版)，1997，18：90-94
[42]潘清，郝文学，发明专利申请公开说明书，CN 1,285，357，1999(20)
[43]郭丽英，朱洪，周元聪，中国生物化学与分子生物学报，1999，15：585-589
[44]Li X.B., Liao, G.S. Shu Y.Y., J. Toxicol. Toxin Rev, 1998, 17:537
[45]Xu T.R., Wang W.Y., Huang Y.H. et al, Comp. Biochem. Physiol. Part C: Pharmacol. Toxicol. Endocrinol, 1999, 724C: 149
[46]奥斯波F．，布伦特R．，金斯顿R．E．，精编分子生物学实验指南，科学出版社，1996，334-338
[47]汪家政，范明，蛋白质技术手册，科学出版社，2000，42-47
[48]Levi-Montalcini R., Meyer H.,Hamburger V., Cancer Res., 1954,14:49
[49]熊仁平，周元国，朱佩芳等，中国生化药物杂志，1996，17：111-113
[50]徐天瑞，董跃伟，熊郁良，药物分析杂志，2000，20：17-19
[51]Siigur J, Arumae U, Siigur E., Recent Advances in Toxinology Research, 1992, 1:154-181
[52]Kostiza T, Dahinden C.A, Rihs S. Otten U., MeierJ., Toxicon, 1995, 33(10):1249-1261
[53]Rukenstein A., Greene L. A., Brain Res., 1983, 263:177
[54]Shaughnessy L.W.,Barone S.J., Neuroreport, 1997, 8:2767
[55]Woo S.B., Timm D.E., Neet K.E., J.Biol. Chem,1995, 270:6278
[56]董跃伟，徐天瑞，熊郁良，云南医药，2000，21：6-8
[57]Guo I.,Leyer S.L.,Kaur H. et.al, Protein Sci.,1996, 5:55
[58]Chevalier S., Praloran V. Smith C. et.al., Blood, 1994, 83:1479-1485
[59]俞萍，赵强，柳川，中国生化药物杂志，1999，20：196-197
[60]郝刚，李文广，高名堂，兰州医学院学报，1998，2
[2]Levi-Montalcini R. et.al, 1951,166:321-362
[3]Cohen S. et.al, Proc. Natl. Acad. Sci. U.S.A., 1954,40:1014-1018
[4]Cohen S. Levi-Montalcini R., Proc. Natl. Acad. Sci. U.S.A., 1956,42:571-574
[5]Levi-Montalcini R., Cohen S., Ibid., 1956,42:695-699
[6]Cohen S.,Symposium Chem.Basis Develop.,Johns Hopkins Univ., 1958:665-676
[7]Cohen S., J. Biol. Chem.,1959, 234:1129-1137
[8]Cohen S., Proc. Natl. Acad. Sci., U.S.A.,1967, 57:1782-1789
[9]Thoenen H., Barde Y.A., Physiol. Rev., 1980, 60:1284
[10]Senut M.C, Lamour Y., J. Lee, P. Brachet, F. Dicou, Neurosecience, 1990, 8:65
[11]Goedert M., Finc A., dawbarn D., Wilcock G.K., Mol. Brain Res., 1989, 5:1
[12]Gibbs R.B., McCabe J.T., Buck C. R, Chao M.V., Pfaff D.W., Mol. Brain Res.,1989, 6:275
[13]Moble W.C., Rutkowski J.L., Jennckoon G.I., Buckanank, Science, 1985, 229:284
[14]Helti F., Neurosci J., 1986, 6:2155
[15]Mark T., James C., Armin B., David S., Pro. Brain Res., 2002, 138:401
[16]Simona C., Sabina G., Antonino C., Mol. Cellular Neurosci., 2002, 21:15
[17]Hellweg H., Hartung H.D., J. Neurosci. Res. 1990, 26:258
[18]李素芹、李法卿、谭维国等，中国生化药物杂志．1999，20：123
[19]祁岩超、周旻、李志阳等，广东医学．1996，17：694
[20]Nishizawa M, Ozawa F, Higashizakit T, Hirai K, Hishinuma F, Appl Microbiol Biotechnol, 1993, 88:624-630
[21]Dicon E, Neurochem Int, 1992, 20:129-134
[22]Lwane M, Biochemi Biophysi Res Commu, 1990, 171:116-122
[23]郭立英，刘杰武，周元聪等，生物化学与生物物理学报，1999，31(2)：203-206
[24]Hogue-Angeletti R.A., J. chromatogr., 1968, 36:535
[25]Hogue-Angeletti R.A., J. chromatogr., 1968, 37:62
[26]Hogue-Angeletti R.A., Frazier W.A., Jacobs, H. J.W. Niall D., Bradshaw R.A., Biochemistry, 1976, 15:26-34
[27]Furukawsa S., Hayashi K., J. Biochem., 1976, 80:1001
[28]Khamidov D.K., Salikhov R.S., Khafizova M.G. et al, Khim. Prir. Soedin., 1985, 4:546
[29]Siigur J., Arumae V., Neuman T. et al, Comp. Biochem. Physiol.,B: Comp.Biochem., 1986, 83B: 621
[30]SiigurG.et.al,Comp.Biochem.Physiol.,B :Comp.Biochem, 1987,87B: 329-334
[31]D. K. Khamidov, L.Y. Ywkelson, R. S.Salikhov et al, Biokhimiya, 1989, 54:987
[32]雷少波，吴秀荣，生物化学与生物物理学报，1989，21：115-119
[33]Smith RJ., Brandt W.F., Stickells B.J. et al, Comp. Biochem. Phusiol., B: Comp. Biochem., 1992, 103B: 975
[34]Kilnion J., Am. Biotechnol. Lab., 1992, 10:18
[35]潘清，郝文学，发明专利申请公开说明书，CN 1，074，448，1992(9)
[36]Koyama J.I., Inoue S., Ikeda K. et al, Biochim. Biophys. Acta, 1992, 1160:787
[37]Horie S., Inoue S., Ikeda K. et al, J. Nat. Toxins, 1994, 3:165
[38]Kostiza T., Dahinden C.A., Rihs S. et al, Toxico., 1995, 33:1249
[39]刘一平，李虹，王锡峰，中国生化药物杂志，1994，15：111-113
[40]刘一平，潘华，李虹等，中国生化药物杂志，1996，17：238-241
[41]刘春宇，陈洁，卢军等，暨南大学学报(自然科学版)，1997，18：90-94
[42]潘清，郝文学，发明专利申请公开说明书，CN 1,285，357，1999(20)
[43]郭丽英，朱洪，周元聪，中国生物化学与分子生物学报，1999，15：585-589
[44]Li X.B., Liao, G.S. Shu Y.Y., J. Toxicol. Toxin Rev, 1998, 17:537
[45]Xu T.R., Wang W.Y., Huang Y.H. et al, Comp. Biochem. Physiol. Part C: Pharmacol. Toxicol. Endocrinol, 1999, 724C: 149
[46]奥斯波F．，布伦特R．，金斯顿R．E．，精编分子生物学实验指南，科学出版社，1996，334-338
[47]汪家政，范明，蛋白质技术手册，科学出版社，2000，42-47
[48]Levi-Montalcini R., Meyer H.,Hamburger V., Cancer Res., 1954,14:49
[49]熊仁平，周元国，朱佩芳等，中国生化药物杂志，1996，17：111-113
[50]徐天瑞，董跃伟，熊郁良，药物分析杂志，2000，20：17-19
[51]Siigur J, Arumae U, Siigur E., Recent Advances in Toxinology Research, 1992, 1:154-181
[52]Kostiza T, Dahinden C.A, Rihs S. Otten U., MeierJ., Toxicon, 1995, 33(10):1249-1261
[53]Rukenstein A., Greene L. A., Brain Res., 1983, 263:177
[54]Shaughnessy L.W.,Barone S.J., Neuroreport, 1997, 8:2767
[55]Woo S.B., Timm D.E., Neet K.E., J.Biol. Chem,1995, 270:6278
[56]董跃伟，徐天瑞，熊郁良，云南医药，2000，21：6-8
[57]Guo I.,Leyer S.L.,Kaur H. et.al, Protein Sci.,1996, 5:55
[58]Chevalier S., Praloran V. Smith C. et.al., Blood, 1994, 83:1479-1485
[59]俞萍，赵强，柳川，中国生化药物杂志，1999，20：196-197
[60]郝刚，李文广，高名堂，兰州医学院学报，1998，2