用噬菌体展示方法研究抗原结合对抗体效应子构象与功能的影响

发布时间：2018-03-04 05:14

本文选题：噬菌体展示技术　切入点：抗原结合　出处：《安徽医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 抗体的Fc段由两条相同免疫球蛋白重链以链间二硫键共价相连构成,可与免疫细胞FcR和C1q补体结合以启动抗体依赖性细胞毒作用(ADCC)以及活化补体级联反应来清除入侵抗原。其中,Fc段与FcR和C1q的结合位点称抗体的效应子(effector),是抗体发挥生物学功能的主要位点。有研究表明,抗体Fc段除了与FcR和C1q结合外,还能与其它的活性蛋白如细菌免疫球蛋白结合蛋白Protein A(金葡菌蛋白A,SpA)和Protein G(链球菌蛋白G,SpG)等结合,而且Fc段与这些不同蛋白的结合位点都集中在一共同的区域即CH2-CH3交界处[1] ,该特定的区域即是抗体效应子的主要位点。抗体上述的两个结构由一个具有高度柔性的连接短肽(铰链区,hinge)相连,使得它们各自保持相对的独立性。抗体与抗原特异结合激发效应子功能的产生是抗体执行其生物学功能的重要途径之一。当抗体与抗原特异结合后,其Fc段与免疫细胞Fc受体、C1q补体的结合得到了启动和加强。为什么结构上相对独立的Fab段特异结合抗原后会激发Fc段与FcR、C1q的结合?两者效应关联的机制是什么?目前得到最多支持和最被接受的解释是:抗体与抗原的特异结合能形成免疫复合物,使得抗体发生聚集,效应子也发生聚集而被激活从而发挥其功能。然而,国外一些学者的研究[2~10]表明还存在另一种可能性,即抗体与抗原特异结合本身能引起抗体效应子构象的改变,从而影响其与免疫细胞Fc受体和C1q补体的结合特性。在众多对抗原结合引发效应子构象改变的研究中,Sagawa等[9]用半抗原与抗体结合改变了其Fc段与Protein A和Protein G结合的报道是最为直接的,有力地证明了在不可能形成抗原抗体聚合物的情况下,抗原抗体结合导致了效应子构象的改变。本研究应用噬菌体展示体外分子进化的方法,以结合抗原与不结合抗原的抗体对同一噬菌体展示免疫球蛋白结合分子组合文库进化筛选,观察所获得的敏感性代表序列是否不同。进一步检测这些序列与抗体结合抗原及不结合抗原时的结合活性,证明抗原结合是否能够引起抗体效应子(Fc段)构象的改变,以及该构象改变对其结合特性的影响,从而阐明抗原结合对其效应功能的影响机制。本研究分以下两部分进行: 一.用兔IgG结合抗原与不结合抗原筛选噬菌体展示免疫球蛋白结合分子组合文库选用包含效应子结合蛋白Protein A的D(PA-D)、A (PA-A)结构域,Protein G的B2结构域(PG)及Protein L的B3结构域(PL)的单结构域随机组合文库,分别应用结合了Tat抗原的兔IgG与未结合Tat抗原的兔IgG,对该组合文库进化筛选,比较分析其进化文库代表性序列的异同性。序列分析后选取代表性阳性单克隆,用ELISA法分别测定其与兔IgG结合抗原及不结合抗原状态下的结合活性,判断不同代表序列与不同状态抗体的结合是否存在倾向性的差别。结果,二者皆进化为天然细菌蛋白中不存在的新的结构形式:在不结合抗原的兔IgG导向的筛选过程中,文库的展示序列全部进化为PA-A—PA-A;而在结合抗原的兔IgG包被抗原的进化文库中全部展示为PA-A—PG结构,二者的代表性序列有显著差异。测定这些不同序列分别与兔IgG结合抗原及不结合抗原的结合活性,结果显示由各自筛选所得的特异分布的序列与相同状态的抗体有着更强的结合反应。二.用鼠IgG结合抗原与不结合抗原筛选噬菌体展示免疫球蛋白结合分子组合文库应用结合了β-HCG抗原的鼠IgG与未结合β-HCG抗原的鼠IgG,对同一噬菌体展示Ig结合分子单结构域组合文库(PALG库)进行高通量体外分子进化筛选,比较分析其进化文库代表性序列的异同性。在不结合抗原的鼠IgG导向的筛选过程中,文库的展示序列大部分都进化为PA-A—PA-A—PG;而在结合抗原的鼠IgG包被抗原的进化中,所展示的序列多种多样,无明显规律性,有待进一步探索。本研究应用结合与不结合抗原的不同动物IgG来研究抗原结合对抗体效应子构象的影响作用,证实了抗原结合确实能够引起抗体效应子构象的改变并可对其结合特性产生影响,有助于阐明抗原结合引发抗体效应功能的一种新的机制。
[Abstract]:Fc fragment consists of two identical immunoglobulin heavy chain to chain two disulfide bonds covalently linked, can be combined with immune cells FcR and C1q complement to start antibody dependent cytotoxicity (ADCC) and activation of the complement cascade to invading antigen binding sites. Among them, Fc and FcR and C1q called effector antibody (effector), is the main site of antibodies play a biological function. Studies have shown that in addition to Fc antibody combined with FcR and C1q, but also with other proteins such as bacterial immunoglobulin binding protein Protein A (staphylococcal protein A, SpA) and Protein G (Streptococcus protein G SpG), combined with these and Fc segment with different protein sites are concentrated in a common area at the junction of CH2-CH3 [1], the specific area is the main site of antibody effector antibody. The two structures mentioned by a highly flexible The connection of short peptides (hinges, hinge) allows them to maintain their respective independence.
The specific binding of antibody and antigen stimulate effector functions of antibody is one of the important ways to perform its biological function. When combined with specific antibody and antigen, the Fc and Fc of immune cells with C1q receptors, complement got started and strengthened. Why the structure is relatively independent of the Fab specific binding of antigen can stimulate Fc FcR and C1q, the combination of the two mechanisms? What is the effect of correlation? Currently get the most support and the most accepted explanation is: the specific antibody and antigen binding to form immune complexes, the antibody aggregation effect also congregate and activated to exert its function. However, some foreign research [2~10] scholars show that there is another possibility that the antibody and antigen specific antibody effector binding itself can cause conformational changes, thus affecting the immune cells and Fc receptor and C1q complement. Photosynthetic characteristics. In many of the antigen binding caused changes of effector conformation in Sagawa [9] with semi antigen antibody combination changed its Fc and Protein A and Protein G combined with the reports is the most direct, effectively proved the formation of antigen antibody polymer in the unlikely case, antigen antibody the combination resulted in a conformational change of the effector.
The research and application of phage display method of molecular evolution in vitro, with the combination of antigen and antibody antigen binding is not on the same phage display library of immunoglobulin binding molecules combined evolutionary selection, whether the different sensitivity of representative sequences was studied. Further detection of these sequences with antibody binding antigen binding activity and antigen, antigen proof whether the combination can cause antibody effector (Fc) conformational change and the conformational change influence the binding properties on it, so as to clarify the mechanism of the effect of antigen binding effect.
This study is divided into two parts as follows:
Screening of phage display immunoglobulin binding library by using rabbit IgG binding antigen and non binding antigen
Involved with the effector binding protein Protein A D (PA-D), A (PA-A) domain, B2 domain Protein G (PG) B3 domain and Protein L (PL) single domain random combinatorial library, were used with Tat antigen in rabbit IgG and unbound Tat antigen in rabbits IgG, the screening of combinatorial library evolution, comparative analysis of the evolution of Library representative sequence similarities and differences. Sequence analysis after selecting the representative positive clones were measured, combined with rabbit IgG antigen binding activity and antigen state by ELISA method. The judge combines different representative sequences with different state whether there is tendency of antibody difference. As a result, the two are for the evolution of new structure does not exist in the natural bacterial protein: in the screening process does not bind to rabbit IgG antigen in the guide, showing all the sequence library evolution of PA-A - PA-A; and in the combination of antigen coated rabbit IgG The evolution of Library antigen in all show PA-A PG structure, the representative of the two sequences with significant differences. The determination of these different sequences were combined with rabbit IgG antigen and not binding activity of the antigen, the results showed that the antibody specific sequence distribution by the respective screening with the same income state has a stronger binding reaction.
Two. Screening of phage display immunoglobulin binding library by using mouse IgG binding antigen and non binding antigen
Application of combination of IgG and beta -HCG antigen were not combined with beta -HCG antigen in IgG, on the same phage display Ig binding molecules single domain combination Library (PALG Library) for high-throughput screening of molecular evolution in vitro, comparative analysis of the evolution of Library representative sequence similarities and differences. In the screening process does not bear in IgG direction combined antigen in the display sequence library most of the evolution of PA-A - PA-A - PG; and in the antigen binding rat IgG antigen in the evolution of a variety of sequence shows diversity, no obvious regularity, needs further exploration.
The research and application of integration with the antigen binding different animal IgG to study the antigen binding effect against effector conformation, confirmed the antigen binding antibody effector can really cause conformational changes and can be combined with the characteristics of its influence, is helpful to elucidate the mechanism of antigen binding a new trigger antibody effector function.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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