Transcript Ch27

Ch27 The Immune System
Yuki Juan
NTU
May 26, 2003
Outline
Introduction
Antibody-based or humoral immunity
Activation of B cells to produce antibodies
T cells and cell-mediated immunity
Why does the human immune system so
fiercely reject foreign human cells
Monoclonal antibodies
Outline
Introduction
Antibody-based or humoral immunity
Activation of B cells to produce antibodies
T cells and cell-mediated immunity
Why does the human immune system so
fiercely reject foreign human cells
Monoclonal antibodies
Terms
Autoimmune reactions:
– The immune system would attack the
components of the body
Autoimmune disease
The Cells Involved In The Immune
System
B cells:
– Multiply and undergo their primary maturation
in bone marrow
T cells:
– Multiple and mature in thymus glands
– Helper T cells and cytotoxic killer T cells
Hemopoiesis
Two Immune Protective Systems
Humoral immunity
Cell-mediated immunity
Humoral Immunity
Antibody:
– Combine with the foreign antigens
Antigen:
– Antibody generation
Cell-mediated Immunity
Cytotoxic T cells or killer T cells recognize
abnormal cells in the body.
The killer cell attaches to the virus-infected
cell and destroys it.
The Differences Between Humoral
And Cell-mediated Immunity
The humoral system:
– is part of the defence against virus in the
blood or in the mucous membrane before it
infects a cell.
Cell-mediated immunity
– Destroys the host cell after the latter has
become infected and in doing so aborts virus
replication in that cell.
Outline
Introduction
Antibody-based or humoral immunity
Activation of B cells to produce antibodies
T cells and cell-mediated immunity
Why does the human immune system so
fiercely reject foreign human cells
Monoclonal antibodies
Antibodies
Immunoglobulins (Igs)
Y-shaped protein
– Two identical light polypeptide chains
– Two identical heavy chains
Structure of Antibody
Terms
Epitope
– The specific part of the antigen which is
recognized by an antibody
Clone
– A collection of identical cells arising from
multiplication of a single cell
Different Classes of Antibodies
IgM
– When the body responds to an antigen, the first
produced antibody
– Multi-subunit or polymeric
– Form of antibody with 10 antigen combining sites
IgG
– More prolonged challenge by the same antigen
IgA
– Of importance as a first of defence
Generation Of Antiboy Diversity
300 V gene fragments
4 J gene segments
C gene segment
The Process of Rearrangment
Leading to a Functional L Chain
Immunoglobulin Gene
Outline
Introduction
Antibody-based or humoral immunity
Activation of B cells to produce antibodies
T cells and cell-mediated immunity
Why does the human immune system so
fiercely reject foreign human cells
Monoclonal antibodies
The Principle of Clonal Selection
Macfarlane
Burnet
Conversion of A
Virgin B Cell to An
Activated State,
But not yet to an
Antibodyproducing Plasma
Cell
MHC classes II
proteins: on the
surface of the B cells
Comparison of Receptors on B and
T Cells
B Cell receptors
T Cell receptors
Are formed from antibodies
displayed in the cell membrane
Special receptor proteins in the
membrane
Specific receptors for each cell
generated randomly
Specific receptors for each cell
generated randomly
Bind to antigen molecules in
solution. Receptor recognizes the
antigen itself
Cannot bind to antigens in solution.
Antigen must be internalized by
special APC which degrades it and
displays peptides in MHC proteins.
The receptor recognises the
complex of peptide-MGC protein
on the APC, not either component
alone
Activation of a Helper T Cell By An
Antigen-presenting Cell (APC)
Cell surface
markers known
as clusters of
differentiation
APC: A special
phagocytic white cell
A Helper T Cell Activating a B Cell
to Become A Clone of Plasma Cells
Secreting Antibody
Summary
Scheme of
Antibody
Production
by B Cells
Term
Memory cells
– They are the basis of long-term immunity from
a repeat infection
Outline
Introduction
Antibody-based or humoral immunity
Activation of B cells to produce antibodies
T cells and cell-mediated immunity
Why does the human immune system so
fiercely reject foreign human cells
Monoclonal antibodies
Class II MHCs and Class I MHCs
Helper T cells
– recognize displayed peptides on class II
MHCs
– Have CD4 proteins
Killer T cells
– require display on class I MHCs
– Have CD8 proteins
Sequence of Events
In Cell-mediated
Immune Reaction to a
Foreign Antigen
Synthesized Inside
Cells
Outline
Introduction
Antibody-based or humoral immunity
Activation of B cells to produce antibodies
T cells and cell-mediated immunity
Why does the human immune system so
fiercely reject foreign human cells
Monoclonal antibodies
Why Does the Human Immune
System So Fiercely Reject Foreign
Human Cells
The main cause of rejection of foreign
tissue grafts in the MHC molecules on
cells.
Outline
Introduction
Antibody-based or humoral immunity
Activation of B cells to produce antibodies
T cells and cell-mediated immunity
Why does the human immune system so
fiercely reject foreign human cells
Monoclonal antibodies
Monoclonal Antibodies
The achievement of this earned Cesar
Milstein and Georges Kohler in Cambridge
a Nobel Prize
單株抗體
由單一細胞株所製造
出來的抗體
多株抗體
抗血清是多株抗體, 這
些抗體是由各種不同
種B細胞所產生抗體之
混合物
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,不
會與不同之抗原決定位置相結合,所以有很
高的專一性
它和抗原決定位置結合的強度及性質完全
相同,所以容易用它來純化抗原
單株抗體的製造方法
正常細胞藉由轉型(transform)變成癌細胞;
也就是使正常細胞表現出致癌基因
(oncogene)
如何使抗體分泌細胞變成癌細胞
– 用致癌物質或病毒使此類細胞內表現致癌基因
– 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融合
瘤細胞(hybridoma)
融合瘤細胞(Hybridoma)之製造
1975, Milstein and Kohler發表1984,諾貝
爾生理醫學獎
老鼠的融合瘤細胞
脾臟B細胞 + 骨髓瘤細胞
 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
自該細胞的組織培養中，收集所需抗體
單株抗體的應用
檢驗試劑的開發製造
醫學治療
生技產品之純化
農業及畜牧業
基礎研究
單株抗體的應用檢驗試劑的開發製造
荷爾蒙
組織及細胞抗原
藥物監控
藥物濫用
微生物
抗體及過敏原
酵素
生長因子
單株抗體的應用醫學治療
器官移植
腫瘤影像
抗癌藥
Ch 29 Gene Cloning,
Recombinant DNA Technology,
Genetic Engineering
Yuki Juan
NTU
May 27, 2003
Outline
The first step-cutting the DNA with
restriction endonucleases
Gene cloning, or how genes are isolated
Isolation of a genomic clone of a human
gene
Cloning a human cDNA
What can be done with the cloned DNA
Knockout mice of gene targeting
Outline
The first step-cutting the DNA with
restriction endonucleases
Gene cloning, or how genes are isolated
Isolation of a genomic clone of a human
gene
Cloning a human cDNA
What can be done with the cloned DNA
Knockout mice of gene targeting
DNases and Restriction Enzymes
DNases
– The digestive enzymes of pancreatic juice
– Cut the phosphodiester bonds
– Cut DNA randomly
Restriction enzymes
– Don’t cut DNA randomly
– Each recognizes a specific short sequence of
bases so as to make a cut at a precision point
in the sequence
基因選殖
– 1970, Smith發現第二類核酸
限切酵素，可以很準確分割
DNA
– 1973, Boyer-Cohen-Chang完
成第一基因選殖的工作
The first to be isolated
from E. coli strain R
Outline
The first step-cutting the DNA with
restriction endonucleases
Gene cloning, or how genes are isolated
Isolation of a genomic clone of a human
gene
Cloning a human cDNA
What can be done with the cloned DNA
Knockout mice of gene targeting
Clone
Refers to multiple identical copies
produced from a single origin
Genomic cloning and cDNA cloning
寄主細胞
蛋白質表現系統主要有
– 大腸桿菌: 操作方便、便宜
缺點:
– 沒有修飾蛋白質的能力
– 無法正確摺疊
– 會產生毒素
– 酵母菌
– 昆蟲細胞: 操作較複雜、成本高
– 哺乳類細胞: 操作較複雜、成本高
Outline
The first step-cutting the DNA with
restriction endonucleases
Gene cloning, or how genes are isolated
Isolation of a genomic clone of a human
gene
Cloning a human cDNA
What can be done with the cloned DNA
Knockout mice of gene targeting
第二類核酸限切酵素
鈍端(blunt end)
黏端(sticky end)
連接酵素(ligase)
Sequence of Steps In Producing in
l Bacteriophage a Recombinant
DNA Library
Illustration of Gene
Cloning Using
Bacteriophage
Lambda Phage as
The Cloning Vehicle
Outline
The first step-cutting the DNA with
restriction endonucleases
Gene cloning, or how genes are isolated
Isolation of a genomic clone of a human
gene
Cloning a human cDNA
What can be done with the cloned DNA
Knockout mice of gene targeting
cDNA
Complementary DNA: the double-stranded
DNA copy of mRNA
Genomic DNA: contains introns
Outline
The first step-cutting the DNA with
restriction endonucleases
Gene cloning, or how genes are isolated
Isolation of a genomic clone of a human
gene
Cloning a human cDNA
What can be done with the cloned DNA
Knockout mice of gene targeting
Transferring a l DNA Insert Into A
Plasmid
The Structures of dATP and ddATP
An Autoradiograph Sequencing Gel
Dideoxy
DNAsequencing
Technique
Photograph of an
Actual
Sequencing
Ladder
DNA定序
Maxam-Gilbert Method
G Reaction
T and C Reaction
– ATTGACTTAGCC
– ATTGACTTA
– ATT
–
–
–
–
–
–
–
–
A and G Reaction
–
–
–
–
–
ATTGACTTAGCC
ATTGACTTA
ATTGACTT
ATTG
ATT
ATTGACTTAGCC
ATTGACTTAGC
ATTGACTTAG
ATTGACT
ATTGAC
ATTGA
AT
A
C Reaction
C
C
G
A
T
T
C
A
G
T
T
–
–
–
–
ATTGACTTAGCC
ATTGACTTAGC
ATTGACTTAG
ATTGA
The Basic Principle Underlying
Amplification of A DNA Section by
PCR
聚合酵素連鎖反應
聚合酵素連鎖反應(1)
聚合酵素連鎖反應(2)
聚合酵素連鎖反應(3)
Terms
Transgenesis:
– Genes can be inserted into animals and
plants to give different phenotypes
Gene therapy
– Involves correction of a gene deficiency by
inserting a normal gene into cells
Outline
The first step-cutting the DNA with
restriction endonucleases
Gene cloning, or how genes are isolated
Isolation of a genomic clone of a human
gene
Cloning a human cDNA
What can be done with the cloned DNA
Knockout mice of gene targeting
Terms
Knockout mice
– Mice in which a specific gene has been functionally
eliminated
Stem cells
– Cells can divide without limit
Embryonic stem cells
– Different from other stem cells and differentiated cells
– They give rise every type of cell in the body
– Totipotent
Procedure For Obtaining Knockout
Mice By Gene-targeted Mutation
One Method of Gene Targeting by
Homolgous Recombination in ES
Cells