标题: 探讨血浆中Haptoglobin: 生理功能及纯化方法
Studies of Plasma Haptoglobin: Physiologic Significance and Purification
作者: 岳中曦
毛仁淡
分子医学与生物工程研究所
关键字: 血红素;hemoglobin;haptoglobin
公开日期: 2006
摘要: 人类haptoglobin分成三种表现型:1-1,2-1和2-2。 先前,我们利用血红素亦或是单株抗体(8B1-3A)所制作之亲合性管柱纯化之Hp仍有大量apoA-Ι所污染。针对不纯的问题,我们发展出一套利用亲合性管柱之新式纯化流程。首先,我们制作及纯化单株抗体抗α次单元(3H8)。血浆通过亲合性层析管柱与抗体结合后,利用含0.12 M NaCl之phosphate缓冲液(pH 7.4)冲洗后,在使用含0.04%十二烷基磺酸钠缓冲液(pH 7.4)再次冲洗结合于管柱上的最主要的杂蛋白质apoA-Ι。最后,以含0.1%十二烷基磺酸钠缓冲液(pH 11)将Hp冲提下来,并以1M Tris-HCl 缓冲液(pH 6.8)中和。纯化之Hp具有高纯度去除apoA-Ι之污染,并且仍具有与血红色结合之生化功能。纯化之Hp三种表现型的纯度皆可到达95%以上。此新式纯化方式具有高效率及高纯度。其设计原理及最佳化过程皆详述于内文中。
猪Hp是一个急性蛋白质,它于血浆中的浓度在发炎或感染时会急速升高。Hp之主要功能为负责与血浆中游离血红素结合以抑制其氧化压力。我们研究台湾兰屿迷你猪(n=43)的Hp表现型,发现它们皆为同型二聚体的结构(β-α-α-β)相近于人类Hp1-1表现型。利用西方墨点法及凝胶过滤管柱分析,我们发现有25%的兰屿迷你猪血浆中具有极低或是没有Hp表现(<0.05 mg/ml)。其cDNA转译成胺基酸之序列与普通家猪(NP_999165)比较分析后发现identity高达99.7%。兰屿迷你猪(n=43)于血浆中之平均浓度为0.21 ± 0.25 mg/ml,明显低于普通家猪0.78 ± 0.45 mg/ml。另外,25%的兰屿迷你猪其血浆Hp浓度低于0.05 mg/ml。再者,具低浓度与具高浓度之迷你猪其血红素新陈代谢速率并无显着差异。迷你猪其心血管系统近似于人类,因此被视为重要临床前实验动物,可用于动脉硬化、心导管及心脏移植方面的试验。然而,迷你猪是否可作为发炎相关疾病之实验动物仍未知。因此,此研究对于兰屿迷你猪作为发炎相关疾病之实验动物提供了完整的参考性价值。
Human haptoglobin (Hp) is classified as three phenotypes: Hp 1-1, 2-1, and 2-2. Previously, we have isolated this protein by affinity columns using either hemoglobin or monoclonal antibody (mAb) prepared against Hp β-chain (clone 8B1-3A). The isolated Hp from both methods, however, contaminates plasma apolipoprotein A-I (apoA-I). In the present report, we developed a novel affinity column procedure using a mAb prepared against α-chain of Hp (clone 3H8) for Hp purification. Plasma was first chromatographed onto the column followed by a normal wash with a buffer containing 0.12 M NaCl, 0.02 M phosphate, pH 7.4 (PBS). The bound proteins were then pre-washed with a 0.04% sodium dodecyl sulfate (SDS)-PBS, pH 7.4, to remove the low-affinity bound apoA-I from Hp. Finally, the bound Hp was eluted with a 0.1% SDS-PBS, pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. As a result, the isolated Hp was devoid of apoA-I and able to retain the biological function by forming an Hp-hemoglobin complex. The homogeneity of each isolated Hp 1-1, 2-1 or 2-2 was greater than 95% with a yield greater than 50%. The procedure described here is significantly improved in time consuming, recovery and purity. The rationale, design, and optimization for each step are described in detail.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT009429501
http://hdl.handle.net/11536/81510
显示于类别:Thesis


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