本文選題：Fc片段　切入點：親和膜　出處：《大連理工大學》2011年碩士論文　論文類型：學位論文

【摘要】：蛋白A(Protien A)是某些金黃色葡萄球菌細胞壁蛋白,根據(jù)其可以和人及一些哺乳動物血清中免疫球蛋白的Fc片段特異性結合的特點,已經(jīng)被應用于抗體的純化和自身免疫性疾病的治療。獲得大量高純度、高生物活性的Protein A或類Protein A功能的小分子是其中的關鍵問題之一,為了達到這一目的,本論文制備了偶聯(lián)有Fc片段的纖維素膜,并研究了利用該親和膜進行重組Protein A純化和篩選分子簇結構仿生小分子親和配基的可行性。主要結果如下： 1.優(yōu)化了通過木瓜酶酶解IgG獲得Fc片段的條件,最優(yōu)條件為37℃,pH 7.5,酶解2 h,酶與底物比為480 U/mg。并利用Protein A親和層析柱從酶解混合物分離純化得到Fc片段。 2.研究了偶聯(lián)Fc片段的纖維素膜的制備過程。確定較優(yōu)的NaIO4氧化條件為pH 5的0.1 mol/L檸檬酸緩沖液,NaIO4濃度為0.2 mol/L,氧化時間控制15 min。依次偶聯(lián)間隔臂己二胺和戊二醛,最后將Fc片段固定到活化后的纖維素膜上,該親和膜Fc片段的固定量為11.75 mg/g。 3.偶聯(lián)Fc片段的纖維素膜對Protein A的靜態(tài)飽和吸附量為8.22 mg/g,動態(tài)吸附容量為6.84 mg/g；該膜對大腸桿菌細胞破碎液中重組Protein A具有良好的選擇性,吸附容量為5.38 mg/g,親和膜重復使用8次,其動態(tài)吸附量基本沒有變化；可以將其用于重組Protein A的分離純化。 4.以固定化抗體Fc片段纖維素膜為篩選平臺,利用殼聚糖作為分子簇的結構基礎,以酪胺為模式分子評價了分子簇結構在小分子親和配基的篩選中所能起到的作用。結果表明,殼聚糖-酪胺同單獨的殼聚糖、酪胺相比,與抗體Fc片段纖維素膜的吸附作用更強,每個固定的Fc分子平均可以吸附31.1個功能基團,是Fc能結合溶液游離態(tài)酪胺分子的11.6倍,驗證了這種篩選模式用于固定化蛋白篩選小分子親和配基的可行性。 總之,以上結果說明抗體Fc片段纖維素膜可以應用于rProtein A的分離純化及仿生的分子簇結構的小分子親和配基的篩選。
[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，

本文編號：1587384