抗体Fc片段纤维素膜的制备及其应用

发布时间：2018-03-09 06:26

  本文选题：Fc片段　切入点：亲和膜　出处：《大连理工大学》2011年硕士论文　论文类型：学位论文

【摘要】：蛋白A(Protien A)是某些金黄色葡萄球菌细胞壁蛋白,根据其可以和人及一些哺乳动物血清中免疫球蛋白的Fc片段特异性结合的特点,已经被应用于抗体的纯化和自身免疫性疾病的治疗。获得大量高纯度、高生物活性的Protein A或类Protein A功能的小分子是其中的关键问题之一,为了达到这一目的,本论文制备了偶联有Fc片段的纤维素膜,并研究了利用该亲和膜进行重组Protein A纯化和筛选分子簇结构仿生小分子亲和配基的可行性。主要结果如下： 1.优化了通过木瓜酶酶解IgG获得Fc片段的条件,最优条件为37℃,pH 7.5,酶解2 h,酶与底物比为480 U/mg。并利用Protein A亲和层析柱从酶解混合物分离纯化得到Fc片段。 2.研究了偶联Fc片段的纤维素膜的制备过程。确定较优的NaIO4氧化条件为pH 5的0.1 mol/L柠檬酸缓冲液,NaIO4浓度为0.2 mol/L,氧化时间控制15 min。依次偶联间隔臂己二胺和戊二醛,最后将Fc片段固定到活化后的纤维素膜上,该亲和膜Fc片段的固定量为11.75 mg/g。 3.偶联Fc片段的纤维素膜对Protein A的静态饱和吸附量为8.22 mg/g,动态吸附容量为6.84 mg/g；该膜对大肠杆菌细胞破碎液中重组Protein A具有良好的选择性,吸附容量为5.38 mg/g,亲和膜重复使用8次,其动态吸附量基本没有变化；可以将其用于重组Protein A的分离纯化。 4.以固定化抗体Fc片段纤维素膜为筛选平台,利用壳聚糖作为分子簇的结构基础,以酪胺为模式分子评价了分子簇结构在小分子亲和配基的筛选中所能起到的作用。结果表明,壳聚糖-酪胺同单独的壳聚糖、酪胺相比,与抗体Fc片段纤维素膜的吸附作用更强,每个固定的Fc分子平均可以吸附31.1个功能基团,是Fc能结合溶液游离态酪胺分子的11.6倍,验证了这种筛选模式用于固定化蛋白筛选小分子亲和配基的可行性。 总之,以上结果说明抗体Fc片段纤维素膜可以应用于rProtein A的分离纯化及仿生的分子簇结构的小分子亲和配基的筛选。
[Abstract]:Protien A) is a cell wall protein of some Staphylococcus aureus, which can specifically bind to FC fragment of immunoglobulin in human and some mammalian serum. It has been applied to the purification of antibodies and the treatment of autoimmune diseases. It is one of the key problems to obtain a large number of small molecules with high purity and high bioactivity of Protein A or Protein A like function, in order to achieve this goal, In this paper, cellulose membrane with FC fragment was prepared, and the feasibility of purification of recombinant Protein A and screening of bionic small molecular affinity ligand with FC fragment were studied. The main results were as follows:. 1. The conditions for obtaining FC fragment by enzymatic hydrolysis of papaya by IgG were optimized. The optimum conditions were as follows: pH 7.5 at 37 鈩，

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