免疫球蛋白(Ig)課件

1、Immunoglobulin , IgNOBEL PRIZES FOR ANTIBODY RESEARCHIg gene rearrangementTonegawa1987Monoclonal AbKoler,Milstein & Jerne1984RadioimmunoassayYalow1977Chemical structure of AbEdelman & Poter1972antitoxinEhrlich & Metchnikoff1908Serum therapyvon Bering19011890: von Behring and Kitasato -ANTIBODIESin t

2、he serum of vaccinated individual bind to pathogensEmil von Behring 1854 - 1917, Nobel Prize in 1901 for demonstrating that circulating antitoxins against diphtheria and tetanus toxins conferred immunity. Discovery of Ab Emil von Behring and Shibasaburo Kitasato took serum from animals infected with

3、 diphtheria and injected it into healthy animals. When these animals were later inoculated with diphtheria they were found to be resistant to infection. We now know this method of conferring infection resistance as passive immunity. This first demonstration of defense against infection was revealed

4、and described as mediated by antitoxin. It was clear to Behring and Kitasato that the antitoxin was specific only for diphtheria, it did not confer any defense against other forms of infection. We now know this antitoxin to be antibodies produced specifically against the diphtheria microbe. Albumin-

5、globin -globin -globin正常血清蛋白電泳圖Two basic definitionsAntibody (Ab)A type of globulin which is produced by plasma cell as a result of the introduction of an antigen and which react specifically with the antigen that stimulated its production. Immunoglobulins (Ig)globulins whose structure or function a

6、re similar to antibody.基本概念A(yù)b抗體是B淋巴細(xì)胞識別抗原后增殖分化為漿細(xì)胞所產(chǎn)生的一種能與相應(yīng)抗原發(fā)生特異性結(jié)合的蛋白質(zhì)。Ig免疫球蛋白指化學(xué)結(jié)構(gòu)或生物學(xué)活性與抗體相類似的糖蛋白。Ig的基本結(jié)構(gòu)（IgG ）四肽鏈結(jié)構(gòu)功能區(qū)絞鏈區(qū)接合鏈分泌片酶解片段1. Heavy & Light ChainsCH1VLCLVHCH2 CH3Hinge RegionCarbohydrateDisulfide bondimmunoglobulin molecule is made up of 4 polypeptide chains. There are two long chains,

7、 called the heavy or H chains which weigh between 50 and 75 kilodaltons and two short chains called light or L chains weighing in at 25 kilodaltons. They are linked together by what are called disulfide bonds to form a Y shape molecule. 1)Heavy chain: composed of about 500 a.a 5 Classes of heavy cha

8、in: , , , , . IgA, IgG, IgM, IgD, IgE2) light chain: composed of about 214 a.atype: ,2. Variable & Constant Regions1) V regionN-terminal 1/2L+1/4(1/5)Hhypervarible region(HVR)(complementarity determining region,CDR): formation of the Ag binding siteFramework region(FR):2) C regionC-terminal 1/2L+3/4

9、(4/5)HCV, CH1, CH2, CH3.VHCH1CH2CH3CH4VLCLFabFcCOONH3+功能區(qū)可變區(qū)： VL，VH恒定區(qū)： CL，CH1， CH2，CH3決定簇互補(bǔ)區(qū)（CDR)3. Hinge regionProperties:1)flexible2) Rich in prolineFunction:1)facilitating the interactionBetween Ag and Ab2) facilitating complement fixation絞鏈區(qū)含有較大量的Pro，具有柔韌性，可折疊FabFcPREBINDING CH1 CH2POSTBINDING(

10、IgM CH3，IgG CH2)IgG Molecule Conformational Changes Induced by Antigen BindingBarricaded C1q-bindingsite Exposed C1q-bindingsite 胃蛋白酶(木瓜蛋白酶)FcpFcF(ab)2Fabpapain:-NH2 terminal side of interchain disulfide bonds in hinge regionFab(Ag-binding Fragment)Fc(crystallizable Fragment):4. Enzymatically genera

11、ted Ab fragmentspapainpepsinpepsin(Fab)2pFc(peptides of Fc) 蛋白酶水解片段-S-S-S-SS-SNNCLHVLCLVHCH1CH2CH3木瓜蛋白酶（papain）2 Fab，1 Fc胃蛋白酶（pepsin）F(ab)2, pFcRodney Porter 1917-1985Nobel Prize in 1972Gerald Edelman 1929 -Nobel Prize in 1972接合鏈 Joining chain連接多體 Ig 的蛋白質(zhì)，存在于IgA 和IgM JSCIgA dimer6. Secretory Piece,S

12、P(1)chemical nature:Polypeptide(70kD)(2)function:. enabling IgA to be transported across mucosal tissues into secretions. . protecting sIgA from being proteolytic attack.chain 7. Ig domainsLight and heavy chains are composed of repeating segments that fold independently to form functional units call

13、ed immunoglobulin(Ig) domainsVLVHCLCH1CH2CH3Domains and functionsVH+VL a Ag- binding siteCH+CLAllotypic marker(IgG)CH2C1q binding(IgG)CH3FcR binding(MC,M,B,NK)(IgM)CH3C1q binding(IgE)CH2+CH3FcR binding(mast,basophil)VHCH1CH2CH3CH4VLCLFabHinge regionFcCOONH3+ 1.A four polypeptide chains basic unit co

14、mposed of two identical light chains and two identical heavy chains ,held by disulfide bonds(二硫鍵）。2.Y-shape structure,symmetric.3. NH2 terminal, -COOH terminal4.variable & constant regions.5.domains: polypeptide chainsfolded by disulfide bonds into globular regions.Varible regionConstant region Summ

15、ary. Isotypes, Allotypes and Idiotypes Ig具有雙重性： 做為抗體能結(jié)合抗原（抗體） 做為大分子蛋白質(zhì)能被其它抗體結(jié)合(抗原)Ag1Ab1（Ag2）Ab2IsotypesdefinitionAntigenic determinants that characterize the classes and subclasses of heavy chains and types and subtypes of light chainslocationIgM (kappa)IgG1 (kappa)同一種屬所有個體的Ig分子均具有的抗原特異性。主要存在于Ig的C區(qū)

16、。AllotypesdefinitionAntigenic determinants specified by allelic forms of the Ig geneslocationIgG1 (kappa)Person 2IgG1 (kappa)Person 1同一種屬不同個體間的Ig分子所具有的不同的抗原特異性。主要存在于重鏈和輕鏈的穩(wěn)定區(qū)，實際上可作為一種遺傳標(biāo)志。IdiotypedefinitionUnique antigenic determinants present on individual antibody molecules .locationAg1epitope1Ab1

17、Ab2Ab3同一個體不同Ig的抗原特異性，主要存在于Ig 的CDR區(qū)。. FUNCTIONS OF ANTIBODY抗體的功能 VHCH1CH2CH3CH4VLCLFabFcCOONH3+C region:1、fixation of complement2、binding cells opsonization mediating ADCC mediateing Hypersensitivity I3.crossing the placenta & mucosal tissues V region: Ag bindingNeutralizing toxin & virusAgglutinatio

18、n microbes,Prevention adhesionVHCH1CH2CH3CH4VLCLFabFcCOONH3+C region:1、激活補(bǔ)體2、Fc段結(jié)合到細(xì)胞表面 調(diào)理作用 介導(dǎo) ADCC 介導(dǎo) Hypersensitivity I3. 穿過胎盤與粘膜組織 V region: 結(jié)合抗原中和毒素和病毒凝集作用Neutralizing toxin & virusAgglutination microbesActivation Complement C1rC1sC1qC1rC1sC1qNo activationActivationC1qr2s2opsonizationOPSONIZATi

19、on FcR antibody-dependent cell-mediated cytotoxicityTarget cellAbEffector cellTarget cellA form of nonspecific cytotoxic cell-mediated reaction in which an Effector kills an antibody-coated target cell,presumably by recognition of the Fc region of the cell-bound antibody through an Fc receptor prese

20、nted on the effector cell (e.g., NK cells,neutrophils,Macrophages).ADCCmediating Hypersensitivity Icrossing the placenta & mucosal tissuesCsT. Properties & Biological Activities of Five Classes of Immunoglobulins五類Ig的特性與功能 IgG1.IgG1, IgG2 and IgG4IgG3Four Subclasses of Human IgG structureGeneral str

21、ucture of the four subclasses of human IgG, which differ in the number and arrangement of the interchain disulfide bonds linking the heavy chains. A notable feature of human IgG3 is its 11 interchain disulfide bonds. 1.Properties IgG is the major serum Ig - 75% of serum Ig is IgGIgG is the major Ig

22、in extravascular spacesThe longest half life(t =23days)d) Placental transfer( IgG2)e) Fixes complement ( IgG4)f) Binds to Fc receptors Macrophages, monocytes,.- Opsonization. NK cell, Macrophages-mediating ADCC SPACoagglutination Test means:Not all subclasses fix equally well; IgG4 does not fix comp

23、lementIgG出生后3個月開始合成，35歲接近成人含量最多 占血清總Ig的 75%產(chǎn)生比較晚，是再次應(yīng)答的主要抗體半衰期最長(t =23天)分布最廣，血管外的主要抗體唯一能通過胎盤的Ig激活補(bǔ)體 IgG13（經(jīng)典），IgG4（替代）結(jié)合細(xì)胞 巨噬細(xì)胞，單核細(xì)胞調(diào)理作用 NK細(xì)胞，巨噬細(xì)胞介導(dǎo)ADCC 結(jié)合SPA協(xié)同凝集實驗Secreted IgM (sIgM): pentamerMembrane-bound IgM(mIgM): monomer J 鏈 Iga Igb Iga IgbIgMstructure 1.Properties the 3rd highest serum Igthe

24、first Ig made by fetus and B cellsintravascular Igd) Fixes complemente) the largest size of moleculef) agglutinating microbes e) Binds to Fc receptors Macrophages, monocytes,.- Opsonization. NK cell, Macrophages-mediating ADCCJ Chain1、是分子量最大的Ig，為五聚體2、半衰期短，約5天左右。若血清中出現(xiàn)IgM，表示有近期感染，可作為傳染病早期診斷的指標(biāo)3、個體發(fā)育及

25、種系發(fā)生過程中出現(xiàn)最早4、抗原刺激后最先產(chǎn)生IgM；5、天然ABO血型抗體多為IgM；6、調(diào)理作用、激活補(bǔ)體作用、殺菌作用 比IgG強(qiáng)。IgMJ ChainIgAstructure J ChainSecretory PieceSecreted IgA (sIgA): dimerserum IgA: monomerIgA dimerproperties:SCJ chain 1)the 2nd highest serum Ig 2)the major Ig in secretions.(secretory IgA,sIgA.5-15g/d)3) sIgA :transferred to the n

26、ewborn through colostrum(初乳） IgA血清中含量第二 (血清型 IgA，serum IgA)主要以分泌型存在.(secretory IgA,sIgA) sIgA通過初乳被動轉(zhuǎn)移給新生兒 唾液, 眼淚, 乳汁, 鼻黏膜, 支氣管分泌液, 生殖 道分泌物, 前列腺液, 小腸黏膜分泌液. SCJ chainIgA dimer粘膜局部抗感染的主要抗體 阻止病原體粘附于粘 膜細(xì)胞中和病毒和外毒素 傷寒沙門氏桿菌, 奈瑟氏淋球菌 脊髓灰質(zhì)炎病毒，流感病毒 抗菌作用 抗病毒作用 抗外毒素作用IgEstructure MonomerExtra domain: CH4CH4proper

27、tiesLeast common serum IgBinds to basophils and mast cells (Does not require Ag binding)Allergic reactionsParasitic infections (Helminths)Binds to Fc receptor on eosinophilsDoes not fix complement Structure of IgE moleculeIgE1)含量最少2)與肥大細(xì)胞和嗜堿性粒細(xì)胞表面的FcR受體結(jié)合, 參與型超敏反應(yīng)與抗寄生蟲感染。親細(xì)胞性抗體:不與Ag結(jié)合，即可與肥大細(xì)胞和嗜堿性粒細(xì)胞

28、表面的FcR牢固結(jié)合3)不能激活補(bǔ)體IgDstructure MonomerTail pieceTail Piece a. on the surface of mature B cell serving as BCR (mature marker of B cell)b. in serum(uncertain Ab activity)properties IgD 分 子 的 結(jié) 構(gòu)*在膜表面的表達(dá)是B 細(xì)胞成熟的主要標(biāo)志；*膜型IgD 作為BCR的組成部分，發(fā)揮信號傳導(dǎo)作用IgD. Ig and TCR Gene Rearrangement 1、GOD ( generation of dive

29、rsity)Susumu TonegamaNobel Prize 1987mRNA RNA轉(zhuǎn)錄本 蛋白質(zhì)多肽鏈基因1基因2基因3基因4 染色體DNA外顯子1內(nèi)含子外顯子2外顯子4外顯子3內(nèi)含子內(nèi)含子 完整基因基 因 的 結(jié) 構(gòu)、 轉(zhuǎn) 錄 與 翻 譯 V V V V V V V V J J J J J K 著絲粒 V V V V V V VPREB1 V V J1 L1 J2 L2 J3 L3 J4 L4 J5 L5 J6 L6 J7 L7 IGLL1 IGLL2 IGLL3Germ line gene organization V V D D D D V V D D D D J J J J M

30、 D G3 G1 EP1 A1 GP G2 G4 E1 A2 95個 IGHV 23個 IGHD 9個 IGHD 9個 IGHJ 11個 IGHC 900 kb 240Kb 22Kb 35Kb 300KbIGH （chromosome 14q32） IGK （chromosome 2p12-p11） 1.85M b0.85M b多于90個 IGKV 5個 IGKJ IGKC 11個 IGKV orphonsIGL （chromosome 22q11） 60個 IGLV 800K b 14K b 60K b 670Kb 30Kb 150Kb 7個 IGLJ 與 7個 IGLC 交替排列 3個 I

31、GLL 基因座 IGH基因群包括95個V（50個是功能基因）、32個D、6個J以及以及9個C基因片段。其中23個D片段位于V片段之間。IGHC基因按照如下順序排列：IGHM、IGHD、IGHG3、IGHG1、IGHA1、IGHG2、IGHG4、IGHE、IGHA2。IGL基因組包括60個V（30個是功能基因，20個為假基因）、7個J基因片段和7個C區(qū)基因（3個假基因）。人IGK基因群包括70個V（約半數(shù)為假基因）、5個J基因片段和1個C區(qū)基因。表7-2 人Ig基因片段* 基因片段Ig基因VDJCIGH95（50）23 9（6） 11（9）IGK90（60）051IGL60（30）07 7（4）

32、 *：表中顯示各基因片段的總數(shù)，括號中為除假基因以及無效基因之外 的功能基因片段數(shù)。1.胚系基因 V1 V2 V3 Vn D1 D2 D3 Dn J1 J2 J3 J4 Cm Cd Cg Ce Ca2.基因重排 V1 V2 D2 J3 J4 Cm Cd Cg Ce Ca3.重排基因 V1 V2 D2 J3 J4 Cm Cd Cg Ce Ca5.mRNA剪切 V2D2J3 Cm Cd V2D2J3 CdCmJ4J46.成熟mRNA V2 D2 J3 Cm V2 D2 J3 Cd L VH Cm7.Igm前體多肽 L VH Cd V2D2J3 J4 Cm Cd4.RNA轉(zhuǎn)錄本 Ig 重 鏈 基 因

33、 重 排 IGHIGKIGLVDJ基因片段的組合VDJV x D x J501263600605300307210IGH/IGK(IGL)3600 x(300+210)=1.6x106Imprecise Joining多樣性至少增加100倍Nucleotide Addition多樣性增加2000倍Potential Variation1010-1011Somatichypermutation突變頻率是其它基因的百萬倍以上。使抗體對抗原的親和力增加100-1000倍。2. Class switchingInitially, all B cells carry IgM specific for an antigen and produce IgM antibody in response to exposure to that antigen. Latter, gene rearrangement pe