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1 生物工程学报 Chin J Biotech 2009, October 25; 25(10): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 生物技术与方法祝骥 1, 易喻 1, 吴银飞 2, 朱克寅 3, 梅建凤 1, 陈建澍 1, 应国清 1, ( ), , : 采用金属螯合亲和层析法, 纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体, 对上样缓冲液的 ph 和离子强度洗脱液种类和洗脱方式进行优化结果表明, 采用降低 ph 分步洗脱时, 最佳上样缓冲液为 ph 8.0, 20 mmol/l PB mol/l NaCl, 抗体在 ph 5.0 被洗脱下来, 抗体回收率 80%, 纯度 85% 采用咪唑浓度梯度洗脱时, 最佳的上样缓冲液为 ph 8.0, 20 mmol/l PB + 5 mmol/l 咪唑, 抗体纯度大于 95%, 回收率 65%; 在上样缓冲液中不添加 NaCl 而添加少量的咪唑, 更有利于抗体分离以上洗脱方式都能较好地保持 mab 的生物学活性, 为该抗体的应用提供了必要的实验基础 : 金属螯合亲和层析, 单克隆抗体, 乙肝核心抗原, 小鼠腹水 1 Purification of anti-hbcag monoclonal antibodies using immobilized metal ion affinity chromatography Ji Zhu 1, Yu Yi 1, Yinfei Wu 2, Keyin Zhu 3, Jianfeng Mei 1, Jianshu Chen 1, and Guoqing Ying 1 1 College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou , China 2 ABON Biopharm (Hangzhou) Co., Ltd, Hangzhou , China 3 BIOLINK Biopharm (Hangzhou) Co., Ltd, Hangzhou , China Abstract: Anti-HBcAg monoclonal antibodies from mouse ascites were purified by using immobilized metal ion affinity chromatography. We optimized the conditions of sample loading and elution. The results showed that when the ph stepwise elution was used, the best solution for sample loading was 20 mmol/l phosphate buffer containing 0.5 mol/l sodium chloride at ph 8.0 and the mab was eluted at ph 5.0. The purity of obtained mab was more than 85% and recovery reached 80%. When the adsorbed proteins were eluted by using gradient elution of an imidazole, the best solution for loading condition was 20 mmol/l phosphate buffer containing 5 mmol/l imidazole at ph 8.0. The purity and recovery of antibody were up to 95%. Keywords: immobilized metal ion affinity chromatography, monoclonal antibody, Hepatitis B core antigen, mouse ascites (IMAC), (Cu 2+ Ni 2+ Co 2+ ), [1], [2-3] Received: April 20, 2009; Accepted: August 17, 2009 Corresponding author: Guoqing Ying. bioph@zjut.edu.cn

2 : 1573, 螯合 mab [4],,,, 1 材料和方法 1.1 材料 His Trap HP 螯合 ( ) (5 ml), (25 mm 16 mm I.D.), (34 μm), (15 μmol Ni 2+ /ml, 6% ), ph (3~12), (5.0 ml/min), (20 ml/min), GE Healthcare ( ) (SDS) (AP) (Tris) (DTT) (TEMED) Bio-Rad ( ); G250: GE healthcare ( ); (Protein Marker) ; ; Q-plus (Millipore, USA) ÄKTA purifier ( 280 nm, GE Healthcare ); JM-250 (Bio-Rad ) Gel Doc 1000 (Bio-Rad ); WFI UV-2000 ( ) DELTA 320 ph (Mettler ); RT-6000 (Rayto ); 0.22 μm MWCO (Millipore ); CR22G (Hitachi ) 1.2 方法 ph, ( r/min 15 min,, 0.22 μm ), ph mmol/l PB mol/l NaCl, His Trap HP 螯合, 5.0 ml/min, 20 mmol/l NaAC-HAC mol/l NaCl(pH ), 5.0 ml/min, NaCl, 0 mol/l 0.5 mol/l 1.0 mol/l 1.5 mol/l 2.0 mol/l NaCl + ph 8.0, 20 mmol/l PB, His Trap HP, 5.0 ml/min, 20 mmol/l NaAC-HAC mol/l NaCl(pH ), 5.0 ml/min, 1.2.3,, 1) ph 8.0, 20 mmol/l PB mol/l NaCl, ph ; 2) ph 8.0, 20 mmol/l PB mol/l NaCl +5 mmol/l, 5~500 mmol/l ; 3) ph 8.0, 20 mmol/l PB+5 mmol/l, 5~500 mmol/l His Trap HP, 5.0 ml/min,, SDS-PAGE 12%, 4%, 10 mol/l, R250,,, IgG [5] [6] ELISA,

1574 ISSN1000-3061 CN11-1998/Q Chin J Biotech October 25, 2009 Vol.25 No.10 IgG, IgG SP2/0, [7] 2 结果 2.1 溶液 ph 对抗体吸附的影响 ph 7.5 8.0 8.5 (20 mmol/l PB + 0.5 mol/l NaCl), 20 mmol/l NaAC-HAC + 0.

0, 20 mmol/l PB ), 20 mmol/l NaAC-HAC + 0.5 mol/l NaCl (ph 6.0 5.5 5.0 4.5 图 2 不同 ph 和 NaCl 浓度上样穿透峰的电泳图 Fig.

0; 4: pass-through peak at ph 8.5; 5: pass-through peak at 0 mol/l NaCl; 6: pass-through peak at 0.5 mol/l NaCl; 7: pass-through peak at 1.0 mol/l NaCl; 8: pass-through peak at 1.

3 1574 ISSN CN /Q Chin J Biotech October 25, 2009 Vol.25 No.10 IgG, IgG SP2/0, [7] 2 结果 2.1 溶液 ph 对抗体吸附的影响 ph (20 mmol/l PB mol/l NaCl), 20 mmol/l NaAC-HAC mol/l NaCl(pH ) : ph ,, ( 2), ph, ph, 1 ph 8.0, 2 ph, 3, ph 8.0,, mab, ph 溶液中 NaCl 浓度对抗体吸附的影响 mol/l NaCl(pH 8.0, 20 mmol/l PB ), 20 mmol/l NaAC-HAC mol/l NaCl (ph 图 2 不同 ph 和 NaCl 浓度上样穿透峰的电泳图 Fig. 2 SDS-PAGE analysis of mab purified by using His Trap HP under different sample ph and NaCl concentration. 1: protein marker; 2: pass-through peak at ph 7.5; 3: pass-through peak at ph 8.0; 4: pass-through peak at ph 8.5; 5: pass-through peak at 0 mol/l NaCl; 6: pass-through peak at 0.5 mol/l NaCl; 7: pass-through peak at 1.0 mol/l NaCl; 8: pass-through peak at 1.5 mol/l NaCl; 9: pass-through peak at 2.0 mol/l NaCl. 图 3 溶液 ph 对抗体吸附的影响 Fig. 3 Effect of different loading ph on the antibody absorption. 图 1 上样缓冲液含 0.5 mol/l NaCl, ph 为 8.0 时的层析图 Fig. 1 Chromatogram of mab by using HisTrap HP (ph 8.0, 0.5 mol/l NaCl). 1: pass-through peak; 2: elution peak of impurities at ph 6.0; 3: elution peak of impurities at ph 5.5; 4: elution peak of mab at ph ) : mol/l NaCl,, NaCl, NaCl,, ( 2) NaCl,, ph 6.0,, 1 NaCl 0.5 mol/l mab, 4 NaCl mab, 4 NaCl 0.5 mol/l, mab

: 1575, 0.5 mol/l NaCl 2.3 洗脱液种类和洗脱方式对抗体洗脱的影响 2.3.1 ph ( ph 8.0, 20 mmol/l PB + 0.5 mol/l NaCl), 20 mmol/l NaAC-HAC + 0.5 mol/l NaCl(pH 6.0 5.5 5.0 4.5 4.0) : ph,, ph 5.5 ph 6.0, ph 5.

4 Effect of different NaCl concentration on the antibody absorption. 图 6 ph 分步洗脱时收集各峰的电泳图 Fig. 6 SDS-PAGE analysis of mab purified by using His Trap HP and ph stepwise elution.

4 : 1575, 0.5 mol/l NaCl 2.3 洗脱液种类和洗脱方式对抗体洗脱的影响 ph ( ph 8.0, 20 mmol/l PB mol/l NaCl), 20 mmol/l NaAC-HAC mol/l NaCl(pH ) : ph,, ph 5.5 ph 6.0, ph 5.0 ( 5),,,, ( 6) ( ) ph 8.0, 20 mmol/l PB mol/l NaCl +5 mmol/l, 5~500 mmol/l, :, 80%, 75% 7, 8 图 4 溶液中 NaCl 浓度对抗体吸附的影响 Fig. 4 Effect of different NaCl concentration on the antibody absorption. 图 6 ph 分步洗脱时收集各峰的电泳图 Fig. 6 SDS-PAGE analysis of mab purified by using His Trap HP and ph stepwise elution. 1: protein marker; 2: pass-through peak at ph 8.0; 3: elution peak two at ph 6.0; 4: elution peak three at ph 5.5; 5: elution peak four containing mab at ph 5.0. 图 5 ph 分步洗脱时的层析图 Fig. 5 Chromatogram of mab by using His Trap HP and ph stepwise elution. 1: pass-through peak; 2: elution peak of impurities at ph 6.0; 3: elution peak of impurities at ph 5.5; 4: elution peak of mab at ph 5.0. 图 7 5~500 mmol/l 咪唑浓度梯度洗脱时的层析图 ( 洗脱液中含 0.5 mol/l NaCl) Fig. 7 Chromatogram of mab by using His Trap HP and imidazole concentration gradient elution of mmol/l containing 0.5 mol/l NaCl. 1: pass-through peak by using imidazole concentration of 5 mmol/l; 2: elution peak by using imidazole concentration gradient elution of mmol/l.

1576 ISSN1000-3061 CN11-1998/Q Chin J Biotech October 25, 2009 Vol.25 No.10 图 8 5~500 mmol/l 咪唑浓度梯度洗脱时收集峰的电泳图 ( 洗脱液中含 0.5 mol/l NaCl) Fig.

1: protein marker; 2: pass-through peak by using imidazole concentration of 5 mmol/l; 3: elution peak by using imidazole concentration gradient elution of 5 500 mmol/l. ph 8.

5 1576 ISSN CN /Q Chin J Biotech October 25, 2009 Vol.25 No.10 图 8 5~500 mmol/l 咪唑浓度梯度洗脱时收集峰的电泳图 ( 洗脱液中含 0.5 mol/l NaCl) Fig. 8 SDS-PAGE analysis of mab purified by using His Trap HP and imidazole concentration gradient elution of mmol/l containing 0.5 mol/l NaCl. 1: protein marker; 2: pass-through peak by using imidazole concentration of 5 mmol/l; 3: elution peak by using imidazole concentration gradient elution of mmol/l. ph 8.0, 20 mmol/l PB+5 mmol/l, 5~500 mmol/l : ph 8.0, 20 mmol/l PB mol/l NaCl+5 mmol/l,, 95%, 65% 9, 10 图 mmol/l 咪唑浓度梯度洗脱时的层析图 ( 洗脱液中不含 NaCl) Fig. 9 Chromatogram of mab by using His Trap HP and imidazole concentration gradient elution of mmol/l. 1: pass-through peak by using imidazole concentration of 5 mmol/l; 2 4: elution peak by using imidazole concentration gradient elution of mmol/l. 图 10 5~500 mmol/l 咪唑浓度梯度洗脱时收集峰的电泳图 ( 洗脱液不含 NaCl) Fig. 10 SDS-PAGE analysis of mab purified by using His Trap HP and imidazole concentration gradient elution of mmol/l. 1: protein marker; 2: pass-through peak by using imidazole concentration of 5 mmol/l; 3: elution peak 2 of Fig. 9; 4: elution peak 3 of Fig. 10; 5: elution peak 4 of Fig 讨论 mab, [8] [9] [10],, (IMAC),, [11-12] mab,, mab, IMAC, : 1), 2) [13],,,,, [14] 150 kd, 50 kd, 25 kd

6 : 1577, 69 kd 80 kd ph,, ph,,, ph, ph 6.0, ph 5.5, ph 5.0,,, ph,,, ph,,, ph ph,, ph 6.0,,, ph 8.0, 20 mmol/l PB mol/l NaCl, ( N ) H +,,, ph, [15-16] ( N ),, ph( ) mab,, mab, ph, mab, 80%, 85% ph (ph 8.0~4.0), ph 8.0, 20 mmol/l PB mol/l NaCl+ 5 mmol/l, 5~500 mmol/l, mab 80%, 75% ph 8.0, 20 mmol/l PB+5 mmol/l, 5~500 mmol/l, 95%,, 65%, 0.5 mol/l NaCl, 0~500 mmol/l, mab, mab, (IMAC),, ph, ph 8.0, 20 mmol/l PB mol/l NaCl, ph 5.0, 80%, 85%, ph 8.0, 20 mmol/l PB + 5 mmol/l, 95%, 65%, NaCl, REFERENCES [1] Jiang W, Graham B, Spiccia L, et al. Protein selectivity with immobilized metal ion tacn sorbents: chromatographic studies with human serum proteins and several other globular proteins. Anal Biochem, 1998, 255(1): [2] Grigoriy SC. Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. J Biochem Biophys Methods, 2001, 49(1): [3] Sun XD, LI HQ, Sui HY, et al. Study on protein separation using immobilized metal ion affinity chromatography. Chin J Biotech, 2000, 16(4): ,,,.., 2000, 16(4): [4] Daniela TB, Olivier P, Mustapha B. Immobilized metal-ion affinity chromatography of human antibodies and their proteolytic fragments. J Chromatogr B, 2004, 808(1): [5] Sambrook J, Russell DW. Molecula Cloning: A Laboratory Manual. 3rd ed. Beijing: Science Press, 2002: J, DW.. 3. :, 2002: [6] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72(1/2): [7] Shen GX, Zhou RL. Experimental Techniques of Modern Immunology. 2nd ed. Wuhan: Hubei Science &

1578 ISSN1000-3061 CN11-1998/Q Chin J Biotech October 25, 2009 Vol.25 No.10 Technology Press, 2002: 154 163.,.. 2. :, 2002: 154 163. [8] Andreas S, Andr e K.

7 1578 ISSN CN /Q Chin J Biotech October 25, 2009 Vol.25 No.10 Technology Press, 2002: ,.. 2. :, 2002: [8] Andreas S, Andr e K. Cation exchange chromatography in antibody purification: ph screening for optimized binding and HCP removal. J Chromatogr B, 2007, 848(1): [9] Raja G, Lu W. Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography. J Chromatogr A, 2006, 1107(1/2): [10] Daisuke E, Ryosuke Y, Kouhei T, et al. Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography. Anal Biochem, 2005, 345(2): [11] Galina T, Blanka H, Miroslav B, et al. Nickel and copper complexes of a chelating methacrylate sorbent in the purification of chitinases and specific immunoglobulin G by immobilized metal ion affinity chromatography. J Chromatogr A, 2003, 983(1/2): [12] Kyriaki G, Penelope CI, Theodore KC. One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography. Protein Expr Purif, 2003, 27(2): [13] Azevedo AM, Rosa PA, Ferreira IF. Integrated process for the purification of antibodies combining aqueous twophase extraction, hydrophobic interaction chromatography and size-exclusion chromatography. J Chromatogr A, 2008, 1213(10): [14] Kucerova Z, Majercakova P. Immobilized-metal-ion afinity chromatography as a tool for the qualitative study of pepsinogen prosphorylation. J Biochem Biophys Methods, 2001, 49(1): [15] Schmit L, Ludwig M, Gaub HE, et al. Ametal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy. Biophys J, 2000, 78(6): [16] Ueda EK, Gout PW, Morgant L. Ni(II) based immobilized metalion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts. J Chromatogr A, 200l, 922(12): 科学出版社科学出版中心生命科学分社新书推介黄东益王英主编年 9 月出版 DNA PCR

基础研究食品科学 2011, Vol. 32, No. 07 17 白球蛋白醇溶蛋白和碱溶蛋白等, 发现白果中的蛋白质主要以清蛋白和球蛋白为主, 其中白果清蛋白具有抗氧化和延缓衰老等作

基础研究食品科学 2011, Vol. 32, No. 07 17 白球蛋白醇溶蛋白和碱溶蛋白等, 发现白果中的蛋白质主要以清蛋白和球蛋白为主, 其中白果清蛋白具有抗氧化和延缓衰老等作 16 李莹莹 1, 吴彩娥 1, *, 杨剑婷 2, 范龚健 1 (1. 南京林业大学森林资源与环境学院, 江苏南京 210037;2. 安徽科技学院食品药品学院, 安徽凤阳 233100) 对白果的主要过敏蛋白进行鉴定, 并对其过敏原性进行分析以 SDS-PAGE 和 Western-Blotting