蛋白A-葡聚糖-Fe4纳米磁珠的制备及对IgG分离纯化

摘要/Abstract

摘要： 蛋白A（Staphylococcal Protein A, 简称蛋白A或SPA）是对IgG有特异性吸附能力的生物配基, 将其偶联到葡聚糖修饰的Fe₃O₄纳米磁珠上, 研究SPA纳米磁珠对IgG的纯化能力。采用高温多元醇法合成不同粒径的纳米磁珠, 研究磁珠粒径对IgG吸附量的影响, 并对磁珠静态载量、吸附时间、可重复性进行研究, 为工业化应用提供理论依据。通过控制反应体系中NaOH的浓度成功地合成了平均粒径从30 nm到200 nm的Fe₃O₄纳米磁珠, 通过吸附量试验证明了磁珠粒径对IgG吸附量有较大影响, 当磁珠平均粒径在101.5 nm时对IgG的吸附量最大, 静态吸附载量为84.85 mg/mL, 亲和常数为3.48×10⁶ M^-1。对磁珠进行5次循环再生试验后, 磁珠的吸附性能没有明显的降低。利用磁珠能高效快速地从人血清中纯化到IgG, 纯度达到93.5%（SDS-PAGE）, 回收率为88.7%。因此, 这种磁性分离基质在IgG纯化中有较大的应用潜力,

关键词: 葡聚糖, 纳米磁珠, 蛋白A, IgG纯化

Abstract: The objective of present research is to prepare magnetic dextran nanoparticles conjugated with Staphylococcal Protein A（SPA-MDPs）for the purification of IgG. The magnetic nanoparticles were prepared by following a high-temperature solution-phase procedure, and the average particle size can be tuned from 30 to 200 nm by simply varying the concentration of NaOH during the reaction. The size of SPA-MDPs nanoparticles has a great influence on their adsorption capacity for IgG. A maximum adsorption of human IgG was calculated to be 84.85 mg/mL with a binding affinity constant of 3.48×10⁶ M^-1 when the average size of SPA-MDPs is about 101.5 nm. The experiment results also demonstrated that the SPA-MDPs nanoparticles can be recycled for 5 times with minor loss of adsorption capacity. Moreover, IgG was purified from human plasma with a purity of 93.5% and recovery of 88.7%. Thus, SPA-MDPs show promising potentials for isolation and purification of IgG.

Key words: Dextran, Magnetic nanoparticles, SPA, IgG purification

任敬钢, 程超, 仲从浩, 张丽, 李荣秀. 蛋白A-葡聚糖-Fe⁴纳米磁珠的制备及对IgG分离纯化[J]. 生物技术通报, 2014, 0(7): 201-208.

Ren Jinggang, Cheng Chao, Zhong Conghao, Zhang li, Li Rongxiu. Fe₃O₄ Magnetic Dextran Nanoparticles Modified with SPA Ligand for IgG Purification[J]. Biotechnology Bulletin, 2014, 0(7): 201-208.

参考文献

[1] Hober S, Nord K, Linhult M. Protein A chromatography for antibody purification[J]. Journal of Chromatography B, 2007, 848（1）：40-47.
[2] Speziale P, Pietrocola G, Rindi S, et al. Structural and functional role of Staphylococcus aureus surface components recognizing adhesive matrix molecules of the host[J]. Future Microbiology, 2009, 4（10）：1337-1352.
[3] Hattum HV, Nathaniel Martin I, Ruijtenbeek R, et al. Development of a microarray detection method for galectin cancer proteins based on ligand binding[J]. Analytical Biochemistry, 2012, 434（13）：99-104.
[4] Chang TM. Sempermeable aqueous microcapsules（“Artificial Cells”）：with emphasis on experiments in an extracorporeal shunt system[J]. ASAIO Journal, 1966, 12（1）：13-19.
[5] Ditsch A, Yin J, Laibinis PE, et al. Ion-exchange purification of proteins using magnetic nanoclusters[J]. Biotechnology Progress, 2006, 22（4）：1153-1162.
[6] Boyer C, Whittaker MR, Bulmus V, et al. The design and utility of polymer-stabilized iron-oxide nanoparticles for nanomedicine applications[J]. NPG Asia Materials, 2010, 2（1）：23-30.
[7] Dias A, Hussain A, Marcos AS, et al. A biotechnological perspective on the application of iron oxide magnetic colloids modified with polysaccharides[J]. Biotechnology Advances, 2011, 29（1）：142-155.
[8] Cao Y, Tian W, Gao S, et al. Immobilization staphylococcal protein A on magnetic cellulose microspheres for IgG affinity purification[J]. Artificial Cells, Blood Substitutes and Biotechnology, 2007, 35（5）：467-480.
[9] Tsushima S. Production of human monoclonal autoantibodies that react with islet-cell-surface antigens by EBV-transformed lymphocytes：new method of preselection of ICSA producing lymphocyte by protein A magnetic microspheres[J]. The Hokkaido Journal of Medical Science, 1988, 63（4）：73-88.
[10] Shinkai M. Functional magnetic particles for medical application[J]. Journal of bioscience and bioengineering, 2002, 94（6）：606-613.
[11] Kaittanis C, Nath S, Perez JM. Rapid nanoparticle-mediated monitoring of bacterial metabolic activity and assessment of antimicrobial susceptibility in blood with magnetic relaxation[J]. PLoS One, 2008, 3（9）：3253-3256.
[12] Kaittanis C, Santra S, Perez JM. Emerging nanotechnology-based strategies for the identification of microbial pathogenesis[J]. Advanced Drug Delivery Reviews, 2010, 62（4）：408-423.
[13] Heeb?ll-Nielsen A, Justesen SF, Thomas OR. Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers[J]. Journal of Biotechnology, 2004, 113（1）：247-262.
[14] Huang X, Zhuang J, Chen D, et al. General strategy for designing functionalized magnetic microspheres for different bioapplications[J]. Langmuir, 2009, 25（19）：11657-11663.
[15] Duan H, Shen Z, Wang X, et al. Preparation of immunomagnetic iron-dextran nanoparticles and application in rapid isolation of E. coli O157：H7 from foods[J]. World Journal of Gastroente-rology, 2005, 11（24）：3660-3664.
[16] Kavaz D, Odabas S, Denkbas EB, et al. A practical methodology for IgG purification via chitosan based magnetic nanoparticles[J]. Digest journal of Nanomaterials and Biostructures, 2012, 7（3）：1165-1177.
[17] Hradil J, Pisarev A, Babi? M, et al. Dextran-modified iron oxide nanoparticles[J]. China Particuology, 2007, 5（1）：162-168.
[18] 李玉珍, 刘学涌, 陈晓凯, 等. 两亲磁性高分子微球与人血清白蛋白的相互作用[J]. 中国药学杂志, 2004, 39（9）：682-684.
[19] Teng S F, Sproule K, Husain A, et al. Affinity chromatography on immobilized “biomimetic” ligands：synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand[J]. Journal of Chromatography B：Biomedical Sciences and Applications, 2000, 740（1）：1-15.
[20] Katsos NE, Labrou NE, Clonis YD. Interaction of L-glutamate oxidase with triazine dyes：selection of ligands for affinity chromatography[J]. Journal of Chromatography B, 2004, 807（2）：277-285.
[21] Albarghouthi M, Fara DA, Saleem M, et al. Immobilization of antibodies on alginate-chitosan beads[J]. International Journal of Pharmaceutics, 2000, 206（1）：23-34.
[22] Deshpande SS. Enzyme immunoassays：from concept to product development［M] . Springer, 1996.
[23] Kong Y. Ligand binding on ladder lattices[J]. Biophysical Chemistry, 1999, 81（1）：7-21.
[24] Gao J, Gu H, Xu B. Multifunctional magnetic nanoparticles：design, synthesis, and biomedical applications[J]. Accounts of Chemical Research, 2009, 42（8）：1097-1107.
[25] Dios AS, Díaz-García ME. Multifunctional nanoparticles：analytical prospects[J]. Analytica Chimica Acta, 2010, 666（1）：1-22.
[26] Füglistaller P. Comparison of immunoglobulin binding capacities and ligand leakage using eight different protein A affinity chromatography matrices[J]. Journal of Immunological Methods, 1989, 124（2）：171-177.

蛋白A-葡聚糖-Fe⁴纳米磁珠的制备及对IgG分离纯化

Fe₃O₄ Magnetic Dextran Nanoparticles Modified with SPA Ligand for IgG Purification

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