近來奈米磁性粒子廣泛受到注目，因為磁性奈米粒子之高比表面積可供更多數量之蛋白質進行固定化反應；其超順磁性之特性可以降低自我聚集之反應；而且利用外加磁場之方式，可從反應混合物中輕易分離出磁性奈米粒子。本研究將含有Z domain之蛋白質固定化於磁性奈米粒子上， Z domain蛋白質之分子量約為19 kDa、它和免疫球蛋白IgG之間具有高親和性之結合。首先以Fe+2以及Fe+3離子進行共沉澱反應來製備磁性奈米粒子。磁性奈米粒子經TEM確認其直徑大約為10奈米；其結晶構造以XRD確認為Fe3O4。以3-aminopropyltriethoxysilane （APES）來處理粒子之表面，可獲得表面覆上氨基之磁性奈米粒子，然後再以N-succinimidyl-3-maleimido propionate (NSMP)將粒子表面之氨基進一步修飾，使磁性奈米粒子表面具有maleimido官能基。此官能基可透過thiol-ether linkage和Z domain蛋白質之cysteine以共價鍵結合。由固定化反應前後溶液中Z domain蛋白質濃度之變化，估算出每個磁性奈米粒子表面可固定大約9個Z domain蛋白質。磁性奈米粒子表面官能基及Z domain蛋白質是以FT-IR鑑定。在Z domain蛋白質固定化後，每一毫克之磁性奈米粒子可吸附大約440微克之豬免疫球蛋白與414微克之兔子免疫球蛋白。將磁性奈米粒子進一步直接加入豬血漿中，亦可以直接純化免疫球蛋白，其純度與以蛋白質A親和性色層分析管柱所純化之免疫球蛋白相似。磁性奈米粒子經過八次重複回收使用後，吸附免疫球蛋白之活性仍可維持於80％以上。此外被磁性奈米粒子所吸附之免疫球蛋白仍然保持吸附抗原的活性。本研究結果顯示，Z domain蛋白質可以Thiol-ether鍵固定於磁性奈米粒子並保持吸附免疫球蛋白之活性。此磁性奈米粒子可以應用於免疫球蛋白之分離純化。

　　Nano-sized magnetic particles recently received considerable attention because they have high specific surface area for immobilization of proteins and ligands, they are superparamagnetic materials which reduce self-aggregation and they can be easily separated from reaction mixture by the application of external magnetic field. In this report, Z domain protein was immobilized on magnetic nanoparticle surface. This protein with molecular weight of 19 kDa can bind immunoglobulin G with high affinity. The magnetic nanoparticles were prepared by coprecipitation of Fe+2 and Fe+3 ions in the presence of base. The size of the nanoparticles was about 10nm as determined by TEM. The crystalline structure of the nanoparticles was Fe3O4 as examined by XRD. The surface of the magnetic nanoparticles was treated with 3-aminopropyltriethoxysilane (APES) to obtain amino-silane coated nanoparticles. The amino functional group on the nanoparticle surface was further derivatized with N-succinimidyl-3-maleimido propionate (NSMP) to obtain nanoparticle with maleimido functional group on its surface. The maleimido group was crosslinked to the thiol group of Z domain proteins through a thiol-ether linkage. The amount of Z domain proteins immobilized on nanoparticles was bout 9 protein molecules on one magnetic nanoparticle.　 Both functional groups and Z domain proteins on nanoparticle surface were confirmed by FT-IR.
The nanoparticle with Z domain proteins (nano-Z) was examined for its affinity to immunoglobulins from pig and rabbit. The weights of pig and rabbit immunoglobulins adsorbed by 1 mg of nano-Z were about 440 μg and 414 μg, respectively. The nano-Z was able to absorb immunoglobulins from pig plasma directly with specificity similar to protein A affinity column. The nano-Z was reusable which retained more than 80% of its original activity after eight repeat usages. Besides, the immunoglobulin absorbed by nano-Z retained its affinity to its antigen. In conclusion, the nano-Z can be prepared by immobilization of Z-domain proteins to the surface of magnetic particles through a thiol-ether linkage and nano-Z can potentially be used in immunoglobulin purification.

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