Ch27 The Immune System

Ch27 The Immune SystemCh27 The Immune System

Yuki JuanYuki Juan

NTUNTU

May 26, 2003 May 26, 2003

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IntroductionIntroduction

Antibody-based or humoral immunityAntibody-based or humoral immunity

Activation of B cells to produce antibodiesActivation of B cells to produce antibodies

T cells and cell-mediated immunityT cells and cell-mediated immunity

Why does the human immune system so fiWhy does the human immune system so fiercely reject foreign human cellsercely reject foreign human cells

Monoclonal antibodiesMonoclonal antibodies

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TermsTerms

Autoimmune reactions: Autoimmune reactions: – The immune system would attack the The immune system would attack the

components of the body components of the body

Autoimmune diseaseAutoimmune disease

The Cells Involved In The Immune The Cells Involved In The Immune SystemSystem

B cells: B cells: – Multiply and undergo their primary maturation Multiply and undergo their primary maturation

in bone marrowin bone marrow

T cells: T cells: – Multiple and mature in thymus glandsMultiple and mature in thymus glands– Helper T cells and cytotoxic killer T cellsHelper T cells and cytotoxic killer T cells

HemopoiesisHemopoiesis

Two Immune Protective SystemsTwo Immune Protective Systems

Humoral immunityHumoral immunity

Cell-mediated immunityCell-mediated immunity

Humoral ImmunityHumoral Immunity

Antibody: Antibody: – Combine with the foreign antigensCombine with the foreign antigens

Antigen: Antigen: – AntiAntibody body gengenerationeration

Cell-mediated ImmunityCell-mediated Immunity

Cytotoxic T cells or killer T cells recognize Cytotoxic T cells or killer T cells recognize abnormal cells in the body.abnormal cells in the body.

The killer cell attaches to the virus-infected The killer cell attaches to the virus-infected cell and destroys it.cell and destroys it.

The Differences Between Humoral The Differences Between Humoral And Cell-mediated ImmunityAnd Cell-mediated Immunity

The humoral system:The humoral system:– is part of the defence against virus in the bloois part of the defence against virus in the bloo

d or in the mucous membrane before it infects d or in the mucous membrane before it infects a cell.a cell.

Cell-mediated immunityCell-mediated immunity– Destroys the host cell after the latter has becoDestroys the host cell after the latter has beco

me infected and in doing so aborts virus replicme infected and in doing so aborts virus replication in that cell.ation in that cell.

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AntibodiesAntibodies

Immunoglobulins (Igs)Immunoglobulins (Igs)

Y-shaped proteinY-shaped protein– Two identical light polypeptide chainsTwo identical light polypeptide chains– Two identical heavy chainsTwo identical heavy chains

Structure of Antibody Structure of Antibody

TermsTerms

EpitopeEpitope– The specific part of the antigen which is recogThe specific part of the antigen which is recog

nized by an antibodynized by an antibody

CloneClone– A collection of identical cells arising from multiA collection of identical cells arising from multi

plication of a single cellplication of a single cell

Different Classes of AntibodiesDifferent Classes of Antibodies

IgMIgM– When the body responds to an antigen, the first produWhen the body responds to an antigen, the first produ

ced antibodyced antibody– Multi-subunit or polymericMulti-subunit or polymeric– Form of antibody with 10 antigen combining sitesForm of antibody with 10 antigen combining sites

IgGIgG– More prolonged challenge by the same antigenMore prolonged challenge by the same antigen

IgAIgA– Of importance as a first of defenceOf importance as a first of defence

Generation Of Antiboy DiversityGeneration Of Antiboy Diversity

300 V gene fragments300 V gene fragments

4 J gene segments4 J gene segments

C gene segmentC gene segment

The Process of Rearrangment LeadThe Process of Rearrangment Leading to a Functional L Chain Immunoing to a Functional L Chain Immuno

globulin Geneglobulin Gene

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The Principle of Clonal SelectionThe Principle of Clonal Selection

Macfarlane Macfarlane BurnetBurnet

Conversion of A Conversion of A Virgin B Cell to An Virgin B Cell to An Activated State, Activated State, But not yet to an But not yet to an

Antibody-Antibody-producing Plasma producing Plasma

Cell Cell MHC classes II MHC classes II proteins: on the proteins: on the surface of the B cellssurface of the B cells

Comparison of Receptors on B and Comparison of Receptors on B and T CellsT Cells

B Cell receptorsB Cell receptors T Cell receptorsT Cell receptors

Are formed from antibodies Are formed from antibodies displayed in the cell membranedisplayed in the cell membrane

Special receptor proteins in the Special receptor proteins in the membranemembrane

Specific receptors for each cell Specific receptors for each cell generated randomlygenerated randomly

Specific receptors for each cell geSpecific receptors for each cell generated randomlynerated randomly

Bind to antigen molecules in Bind to antigen molecules in solution. Receptor recognizes the solution. Receptor recognizes the antigen itselfantigen itself

Cannot bind to antigens in solution.Cannot bind to antigens in solution. Antigen must be internalized by s Antigen must be internalized by special APC which degrades it and pecial APC which degrades it and displays peptides in MHC proteins. displays peptides in MHC proteins. The receptor recognises the complThe receptor recognises the complex of peptide-MGC protein on the ex of peptide-MGC protein on the APC, not either component aloneAPC, not either component alone

Activation of a Helper T Cell By An Activation of a Helper T Cell By An Antigen-presenting Cell (APC)Antigen-presenting Cell (APC)

APC: A special phagocytic white cell

Cell surface markers known as clusters of differentiation

A Helper T Cell Activating a B Cell A Helper T Cell Activating a B Cell to Become A Clone of Plasma Cells to Become A Clone of Plasma Cells

Secreting AntibodySecreting Antibody

Summary Summary Scheme of Scheme of Antibody Antibody

Production Production by B Cellsby B Cells

TermTerm

Memory cellsMemory cells– They are the basis of long-term immunity from They are the basis of long-term immunity from

a repeat infectiona repeat infection

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Class II MHCs and Class I MHCsClass II MHCs and Class I MHCs

Helper T cells Helper T cells – recognize displayed peptides on class II MHCrecognize displayed peptides on class II MHC

ss– Have CD4 proteinsHave CD4 proteins

Killer T cells Killer T cells – require display on class I MHCsrequire display on class I MHCs– Have CD8 proteinsHave CD8 proteins

Sequence of Events Sequence of Events In Cell-mediated In Cell-mediated

Immune Reaction to a Immune Reaction to a Foreign Antigen Foreign Antigen

Synthesized Inside Synthesized Inside CellsCells

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Why Does the Human Immune SystWhy Does the Human Immune System So Fiercely Reject Foreign Humem So Fiercely Reject Foreign Hum

an Cellsan Cells

The main cause of rejection of foreign The main cause of rejection of foreign tissue grafts in the MHC molecules on tissue grafts in the MHC molecules on cells. cells.

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Monoclonal AntibodiesMonoclonal Antibodies

The achievement of this earned Cesar The achievement of this earned Cesar Milstein and Georges Kohler in Cambridge Milstein and Georges Kohler in Cambridge a Nobel Prizea Nobel Prize

單株抗體單株抗體由單一細胞株所製造由單一細胞株所製造出來的抗體出來的抗體

多株抗體多株抗體抗血清是多株抗體抗血清是多株抗體 , , 這些抗體是由各種不這些抗體是由各種不同種同種 BB 細胞所產生抗細胞所產生抗體之混合物體之混合物

單株抗體的特株性質單株抗體的特株性質它只和抗原中的某種抗原決定位置結合它只和抗原中的某種抗原決定位置結合 ,, 不不會與不同之抗原決定位置相結合會與不同之抗原決定位置相結合 ,, 所以有很所以有很高的專一性高的專一性它和抗原決定位置結合的強度及性質完全它和抗原決定位置結合的強度及性質完全相同相同 ,, 所以容易用它來純化抗原所以容易用它來純化抗原

單株抗體的製造方法單株抗體的製造方法正常細胞正常細胞藉由轉型藉由轉型 (transform)(transform) 變成變成癌細胞癌細胞 ;;也就是使正常細胞表現出致癌基因也就是使正常細胞表現出致癌基因 (oncoge(oncogene)ne)

如何使抗體分泌細胞變成癌細胞如何使抗體分泌細胞變成癌細胞– 用用致癌物質致癌物質或或病毒病毒使此類細胞內表現致癌基因使此類細胞內表現致癌基因– 細胞融合技術細胞融合技術

致癌細胞之致癌基因融合到抗體產分泌細胞中致癌細胞之致癌基因融合到抗體產分泌細胞中融合融合瘤細胞瘤細胞 (hybridoma)(hybridoma)

融合瘤細胞融合瘤細胞 (Hybridoma)(Hybridoma) 之製造之製造1975, Milstein and Kohler1975, Milstein and Kohler 發表發表 1984,1984, 諾諾貝爾生理醫學獎貝爾生理醫學獎老鼠的融合瘤細胞老鼠的融合瘤細胞脾臟脾臟 BB 細胞細胞 + + 骨髓瘤細胞骨髓瘤細胞以特殊溶液處理，分離融合細胞以特殊溶液處理，分離融合細胞篩選具有特殊抗體的細胞株篩選具有特殊抗體的細胞株自該細胞的組織培養中，收集所需抗體自該細胞的組織培養中，收集所需抗體

單株抗體的應用單株抗體的應用檢驗試劑的開發製造檢驗試劑的開發製造醫學治療醫學治療生技產品之純化生技產品之純化農業及畜牧業農業及畜牧業基礎研究基礎研究

單株抗體的應用單株抗體的應用 --檢驗試劑的開發製造檢驗試劑的開發製造

荷爾蒙荷爾蒙組織及細胞抗原組織及細胞抗原藥物監控藥物監控藥物濫用藥物濫用微生物微生物抗體及過敏原抗體及過敏原酵素酵素生長因子生長因子

單株抗體的應用單株抗體的應用 --醫學治療醫學治療

器官移植器官移植腫瘤影像腫瘤影像抗癌藥抗癌藥

Ch 29 Gene Cloning, Ch 29 Gene Cloning, Recombinant DNA Technology, Recombinant DNA Technology,

Genetic EngineeringGenetic Engineering

Yuki JuanYuki Juan

NTUNTU

May 27, 2003May 27, 2003

OutlineOutline

The first step-cutting the DNA with restrictiThe first step-cutting the DNA with restriction endonucleaseson endonucleases

Gene cloning, or how genes are isolatedGene cloning, or how genes are isolated

Isolation of a genomic clone of a human gIsolation of a genomic clone of a human geneene

Cloning a human cDNACloning a human cDNA

What can be done with the cloned DNAWhat can be done with the cloned DNA

Knockout mice of gene targetingKnockout mice of gene targeting

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DNases and Restriction EnzymesDNases and Restriction Enzymes

DNasesDNases– The digestive enzymes of pancreatic juiceThe digestive enzymes of pancreatic juice– Cut the phosphodiester bondsCut the phosphodiester bonds– Cut DNA randomlyCut DNA randomly

Restriction enzymesRestriction enzymes– Don’t cut DNA randomlyDon’t cut DNA randomly– Each recognizes a specific short sequence of Each recognizes a specific short sequence of

bases so as to make a cut at a precision point bases so as to make a cut at a precision point in the sequence in the sequence

基因選殖基因選殖– 1970, Smith1970, Smith 發現第二類核酸發現第二類核酸

限切酵素限切酵素，，可以很準確分割可以很準確分割 DDNANA

– 1973, Boyer-Cohen-Chang1973, Boyer-Cohen-Chang完成第一基因選殖的工作完成第一基因選殖的工作

The first to be isolated from E. coli strain R

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CloneClone

Refers to multiple identical copies produceRefers to multiple identical copies produced from a single origind from a single origin

Genomic cloning and cDNA cloningGenomic cloning and cDNA cloning

寄主細胞寄主細胞蛋白質表現系統主要有蛋白質表現系統主要有– 大腸桿菌大腸桿菌 : : 操作方便、便宜操作方便、便宜

缺點缺點 : : – 沒有修飾蛋白質的能力沒有修飾蛋白質的能力– 無法正確摺疊無法正確摺疊 – 會產生毒素會產生毒素

– 酵母菌酵母菌– 昆蟲細胞昆蟲細胞 : : 操作較複雜、成本高操作較複雜、成本高– 哺乳類細胞哺乳類細胞 : : 操作較複雜、成本操作較複雜、成本

高高

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第二類核酸限切酵素第二類核酸限切酵素

鈍端 (blunt end)黏端 (sticky end)

連接酵素連接酵素 (ligase)(ligase)

Sequence of Steps In Producing in Sequence of Steps In Producing in Bacteriophage a Recombinant DBacteriophage a Recombinant D

NA LibraryNA Library

Illustration of Gene CIllustration of Gene Cloning Using Bacterioloning Using Bacteriophage Lambda Phagphage Lambda Phage as The Cloning Vee as The Cloning Ve

hiclehicle

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cDNAcDNA

Complementary DNA: the double-stranded Complementary DNA: the double-stranded DNA copy of mRNADNA copy of mRNA

Genomic DNA: contains intronsGenomic DNA: contains introns

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Transferring a Transferring a DNA Insert Into A DNA Insert Into A PlasmidPlasmid

The Structures of dATP and ddATPThe Structures of dATP and ddATP

An Autoradiograph Sequencing GelAn Autoradiograph Sequencing Gel

Dideoxy DNDideoxy DNA-sequenciA-sequencing Techniqng Techniqueue

Photograph of an Photograph of an Actual Actual

Sequencing Sequencing LadderLadder

DNADNA 定序定序Maxam-Gilbert MethodMaxam-Gilbert Method

G ReactionG Reaction– ATTGACTTAGCCATTGACTTAGCC– ATTGACTTAATTGACTTA– ATTATT

A and G ReactionA and G Reaction– ATTGACTTAGCCATTGACTTAGCC– ATTGACTTAATTGACTTA– ATTGACTTATTGACTT– ATTGATTG– ATTATT

T and C ReactionT and C Reaction– ATTGACTTAGCCATTGACTTAGCC– ATTGACTTAGCATTGACTTAGC– ATTGACTTAGATTGACTTAG– ATTGACTATTGACT– ATTGACATTGAC– ATTGAATTGA– ATAT– AA

C ReactionC Reaction– ATTGACTTAGCCATTGACTTAGCC– ATTGACTTAGCATTGACTTAGC– ATTGACTTAGATTGACTTAG– ATTGAATTGA

CCGATTCAGTT

The Basic Principle Underlying The Basic Principle Underlying Amplification of A DNA Section by Amplification of A DNA Section by

PCRPCR

聚合酵素連鎖反應聚合酵素連鎖反應

聚合酵素連鎖反應聚合酵素連鎖反應 (1)(1)



TermsTerms

Transgenesis:Transgenesis:– Genes can be inserted into animals and plantGenes can be inserted into animals and plant

s to give different phenotypess to give different phenotypes

Gene therapyGene therapy– Involves correction of a gene deficiency by insInvolves correction of a gene deficiency by ins

erting a normal gene into cellserting a normal gene into cells

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TermsTerms

Knockout miceKnockout mice– Mice in which a specific gene has been functionally eliMice in which a specific gene has been functionally eli

minated minated

Stem cellsStem cells– Cells can divide without limitCells can divide without limit

Embryonic stem cellsEmbryonic stem cells– Different from other stem cells and differentiated cellsDifferent from other stem cells and differentiated cells– They give rise every type of cell in the bodyThey give rise every type of cell in the body– TotipotentTotipotent

Procedure For Obtaining Knockout Procedure For Obtaining Knockout Mice By Gene-targeted MutationMice By Gene-targeted Mutation

One Method of Gene Targeting by One Method of Gene Targeting by Homolgous Recombination in ES CHomolgous Recombination in ES C

ellsells

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Ch27 The Immune System