羟基磷灰石纳米微粒的制备及其纯化组氨酸融合蛋白

发布时间：2018-04-20 23:10

本文选题：纳米微粒 + 亲和分离　；参考：《河南大学》2015年硕士论文

【摘要】：本文利用液相化学法制备了HAP/IDA-Ni2+、Fe3O4/HAP-Ni2+、Ni(OH)2/HAP及Ni(OH)2/HAP-RBH纳米微粒,并用多种方法对合成微粒进行表征。利用Ni2+与组氨酸标签(His-tagged)蛋白质之间的亲和作用,将这些纳米微粒作为吸附剂实现了对His-tagged蛋白质的分离与纯化,并对其亲和分离行为进行了研究。具体研究内容和结论如下:(1)HAP/IDA纳米微粒的制备及分离纯化His-tagged蛋白质采用水热法在SBF溶液中成功制备了多孔的HAP纳米微粒,平均粒径在40 nm左右。利用HAP纳米微粒表面的活性羟基,成功实现了其表面IDA功能化,进一步螯合Ni2+后,得到HAP/IDA-Ni2+纳米微粒。这种纳米微粒能够从蛋白混合液中一步纯化His-tagged蛋白质。结果表明,HAP/IDA-Ni2+纳米微粒表现出高的特异性和吸附能力,重复利用四次后,材料依然保持较高的分离性能。HAP/IDA-Ni2+纳米微粒适用于分离低分子量His-tagged蛋白质。(2)Fe3O4/HAP纳米微粒的制备及分离纯化His-tagged蛋白质采用水热法成功制备了具有磁响应性能的Fe3O4/HAP纳米微粒。进一步螯合Ni2+后,Fe3O4/HAP-Ni2+纳米微粒在外加磁场条件下可以直接从蛋白混合液中纯化His-tagged蛋白质。结果表明,该纳米微粒对His-tagged蛋白质具有高的选择性和相对弱的非特异性吸附,其最大吸附量为12.98 mmol/g。(3)Ni(OH)2/HAP纳米微粒的制备及分离纯化His-tagged蛋白质采用水热法成功制备了Ni(OH)2/HAP纳米微粒,并考察了反应条件和Ni2+浓度对Ni(OH)2/HAP粒径和形貌的影响,Ni2+浓度越低,所得纳米微粒的粒径则越小。这种纳米微粒可以直接用于纯化His-tagged蛋白质,并采用三种His-tagged蛋白质对其亲和特性进行了评价。结果表明,Ni(OH)2/HAP纳米微粒对His-tagged蛋白质具有很高的亲和分离特性和对His-tag的普适性。(4)Ni(OH)2/HAP-RBH纳米微粒的制备及分离纯化His-tagged蛋白质采用三步法成功制备了多功能Ni(OH)2/HAP-RBH纳米微粒。首先在柠檬酸存在下,水热法合成Ni(OH)2/HAP纳米微粒。其次罗丹明B与水合肼回流12 h合成罗丹明酰肼(RBH)。最后RBH与微粒表面羧基反应形成Ni(OH)2/HAP-RBH纳米微粒。作为亲和吸附剂,该粒子可以用于直接净化His-tagged蛋白质,并表现出极好的蛋白质吸附能力,最高吸附量为115 mg/g。Ni(OH)2/HAP-RBH纳米微粒对Pt2+表现出良好的荧光增强效应、灵敏性和选择性,检出限为3.0×10-8 mol/L,可作为Pt2+探针用于荧光成像。
[Abstract]:In this paper, HAP/IDA-Ni2 Fe3O4 / HAP-Ni2 and Ni(OH)2/HAP-RBH nanoparticles were prepared by liquid-phase chemical method and characterized by various methods. Based on the affinity between Ni2 and His-tagged protein, the His-tagged protein was separated and purified by using these nanoparticles as adsorbents, and the affinity separation behavior was studied. The specific research contents and conclusions are as follows: the preparation and purification of His-tagged protein by His-tagged nanoparticles were studied. The porous HAP nanoparticles were successfully prepared by hydrothermal method in SBF solution. The average diameter of HAP nanoparticles was about 40 nm. The IDA functionalization of HAP nanoparticles was successfully realized by using the active hydroxyl groups on the surface of HAP nanoparticles. After chelating Ni2 further, HAP/IDA-Ni2 nanoparticles were obtained. The nanoparticles can purify His-tagged proteins from protein mixtures in one step. The results showed that HAP / IDA-Ni2 nanoparticles showed high specificity and adsorption ability, and were reused for four times. HAP / IDA-Ni2 nanoparticles were suitable for the preparation of low molecular weight His-tagged protein, Fe3O4 / HAP nanoparticles and the preparation of purified His-tagged proteins. The magnetically responsive Fe3O4/HAP nanoparticles were successfully prepared by hydrothermal method. After further chelating Ni2, Fe _ 3O _ 4 / HAP-Ni _ 2 nanoparticles can purify His-tagged protein directly from protein mixture under the condition of external magnetic field. The results showed that the nanoparticles had high selectivity and relatively weak nonspecific adsorption on His-tagged protein, and the maximum adsorption capacity was 12.98 mmol/g.(3)Ni(OH)2/HAP nanoparticles. The preparation and purification of His-tagged protein were successfully prepared by hydrothermal method. The effects of reaction conditions and Ni2 concentration on the particle size and morphology of Ni(OH)2/HAP were investigated. The smaller the concentration of Ni2 was, the smaller the particle size was. The nanoparticles can be directly used to purify His-tagged proteins and their affinity properties are evaluated by using three His-tagged proteins. The results showed that the two HAP nanoparticles had high affinity for His-tagged protein separation, and the preparation and purification of His-tagged protein by His-tag. The preparation and purification of His-tagged nanoparticles were successfully carried out by three-step method. Firstly, Ni(OH)2/HAP nanoparticles were synthesized by hydrothermal method in the presence of citric acid. Secondly, Rhodamine B was refluxed with hydrazine hydrate for 12 h to synthesize Rhodamine acyl hydrazide (RBH). Finally, RBH reacts with carboxyl group on the surface of particles to form Ni(OH)2/HAP-RBH nanoparticles. As an affinity adsorbent, the particle can be used to purify His-tagged protein directly and exhibit excellent protein adsorption ability. The maximum adsorption capacity of the nanoparticles is 115 mg/g.Ni(OH)2/HAP-RBH nanoparticles with good fluorescence enhancement effect, sensitivity and selectivity to Pt2. The detection limit is 3.0 脳 10 ~ (-8) mol / L, which can be used as a Pt2 probe for fluorescence imaging.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1

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