本文選題：噬菌體展示技術(shù)　切入點：抗原結(jié)合　出處：《安徽醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 抗體的Fc段由兩條相同免疫球蛋白重鏈以鏈間二硫鍵共價相連構(gòu)成,可與免疫細胞FcR和C1q補體結(jié)合以啟動抗體依賴性細胞毒作用(ADCC)以及活化補體級聯(lián)反應(yīng)來清除入侵抗原。其中,Fc段與FcR和C1q的結(jié)合位點稱抗體的效應(yīng)子(effector),是抗體發(fā)揮生物學(xué)功能的主要位點。有研究表明,抗體Fc段除了與FcR和C1q結(jié)合外,還能與其它的活性蛋白如細菌免疫球蛋白結(jié)合蛋白Protein A(金葡菌蛋白A,SpA)和Protein G(鏈球菌蛋白G,SpG)等結(jié)合,而且Fc段與這些不同蛋白的結(jié)合位點都集中在一共同的區(qū)域即CH2-CH3交界處[1] ,該特定的區(qū)域即是抗體效應(yīng)子的主要位點。抗體上述的兩個結(jié)構(gòu)由一個具有高度柔性的連接短肽(鉸鏈區(qū),hinge)相連,使得它們各自保持相對的獨立性。 抗體與抗原特異結(jié)合激發(fā)效應(yīng)子功能的產(chǎn)生是抗體執(zhí)行其生物學(xué)功能的重要途徑之一。當抗體與抗原特異結(jié)合后,其Fc段與免疫細胞Fc受體、C1q補體的結(jié)合得到了啟動和加強。為什么結(jié)構(gòu)上相對獨立的Fab段特異結(jié)合抗原后會激發(fā)Fc段與FcR、C1q的結(jié)合?兩者效應(yīng)關(guān)聯(lián)的機制是什么?目前得到最多支持和最被接受的解釋是:抗體與抗原的特異結(jié)合能形成免疫復(fù)合物,使得抗體發(fā)生聚集,效應(yīng)子也發(fā)生聚集而被激活從而發(fā)揮其功能。然而,國外一些學(xué)者的研究[2~10]表明還存在另一種可能性,即抗體與抗原特異結(jié)合本身能引起抗體效應(yīng)子構(gòu)象的改變,從而影響其與免疫細胞Fc受體和C1q補體的結(jié)合特性。在眾多對抗原結(jié)合引發(fā)效應(yīng)子構(gòu)象改變的研究中,Sagawa等[9]用半抗原與抗體結(jié)合改變了其Fc段與Protein A和Protein G結(jié)合的報道是最為直接的,有力地證明了在不可能形成抗原抗體聚合物的情況下,抗原抗體結(jié)合導(dǎo)致了效應(yīng)子構(gòu)象的改變。 本研究應(yīng)用噬菌體展示體外分子進化的方法,以結(jié)合抗原與不結(jié)合抗原的抗體對同一噬菌體展示免疫球蛋白結(jié)合分子組合文庫進化篩選,觀察所獲得的敏感性代表序列是否不同。進一步檢測這些序列與抗體結(jié)合抗原及不結(jié)合抗原時的結(jié)合活性,證明抗原結(jié)合是否能夠引起抗體效應(yīng)子(Fc段)構(gòu)象的改變,以及該構(gòu)象改變對其結(jié)合特性的影響,從而闡明抗原結(jié)合對其效應(yīng)功能的影響機制。 本研究分以下兩部分進行: 一.用兔IgG結(jié)合抗原與不結(jié)合抗原篩選噬菌體展示免疫球蛋白結(jié)合分子組合文庫 選用包含效應(yīng)子結(jié)合蛋白Protein A的D(PA-D)、A (PA-A)結(jié)構(gòu)域,Protein G的B2結(jié)構(gòu)域(PG)及Protein L的B3結(jié)構(gòu)域(PL)的單結(jié)構(gòu)域隨機組合文庫,分別應(yīng)用結(jié)合了Tat抗原的兔IgG與未結(jié)合Tat抗原的兔IgG,對該組合文庫進化篩選,比較分析其進化文庫代表性序列的異同性。序列分析后選取代表性陽性單克隆,用ELISA法分別測定其與兔IgG結(jié)合抗原及不結(jié)合抗原狀態(tài)下的結(jié)合活性,判斷不同代表序列與不同狀態(tài)抗體的結(jié)合是否存在傾向性的差別。結(jié)果,二者皆進化為天然細菌蛋白中不存在的新的結(jié)構(gòu)形式:在不結(jié)合抗原的兔IgG導(dǎo)向的篩選過程中,文庫的展示序列全部進化為PA-A—PA-A;而在結(jié)合抗原的兔IgG包被抗原的進化文庫中全部展示為PA-A—PG結(jié)構(gòu),二者的代表性序列有顯著差異。測定這些不同序列分別與兔IgG結(jié)合抗原及不結(jié)合抗原的結(jié)合活性,結(jié)果顯示由各自篩選所得的特異分布的序列與相同狀態(tài)的抗體有著更強的結(jié)合反應(yīng)。 二.用鼠IgG結(jié)合抗原與不結(jié)合抗原篩選噬菌體展示免疫球蛋白結(jié)合分子組合文庫 應(yīng)用結(jié)合了β-HCG抗原的鼠IgG與未結(jié)合β-HCG抗原的鼠IgG,對同一噬菌體展示Ig結(jié)合分子單結(jié)構(gòu)域組合文庫(PALG庫)進行高通量體外分子進化篩選,比較分析其進化文庫代表性序列的異同性。在不結(jié)合抗原的鼠IgG導(dǎo)向的篩選過程中,文庫的展示序列大部分都進化為PA-A—PA-A—PG;而在結(jié)合抗原的鼠IgG包被抗原的進化中,所展示的序列多種多樣,無明顯規(guī)律性,有待進一步探索。 本研究應(yīng)用結(jié)合與不結(jié)合抗原的不同動物IgG來研究抗原結(jié)合對抗體效應(yīng)子構(gòu)象的影響作用,證實了抗原結(jié)合確實能夠引起抗體效應(yīng)子構(gòu)象的改變并可對其結(jié)合特性產(chǎn)生影響,有助于闡明抗原結(jié)合引發(fā)抗體效應(yīng)功能的一種新的機制。
[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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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本文編號：1564273