Download Cell and Enzyme Immobilization

Introduction
to
Biological Science
By: Chung W.Wu,M.S.,R.Ph
Ext:---5213
生物科技概論
Ancient Biological Science
1.Food Preparation
Beer,Cheese
2.Fermentation
Microbial
Food--------------Perfume
3.Alcohol
Fermentation
Distillation
Alcohol Concentration
1st World War
Germany
Glucose---------Glycerol
British
Sugar---------------------Acetone+Butanol
Clostridium acetobutylicum
Carbon Dioxide
Hydrogen Gas:Explosive
2nd World War
1940 Fleming
Penicillium notatum
Penicillin:
Broad Spectrum Antibiotic
2mg/l
Penicillium chrysogenum
UV,X-Ray—Mutation
20mg/l
Streptomyces griseus
Stretomycin
Genetic Engineering
DNA Recombination
Recombinant
Deoxyribonucleic Acid
Chemical Composition
1. Phosphate
2. Pentose
3. Nitrogenous Base
Adenine
Thymine
Cytosine
Guanine
Principles of DNA Recombinant
DNA Section can’t Replicate by itself.
1. Cloning Vector
具有複製起始能力之載體
2. Plasmid
能獨立複製之環狀 DNA 分子
3. Restriction Endonuclease
(DNase)
Recognition Site
4. Sticky Ends
Expression of Cloned Genes
1. Transcription
2. Translation
3. Post-Translation
Modification
The Molecular Basis of
Heredity: DNA & RNA
Nuclear Components
DNA→Transcription→RNA
→Translation→Amino Acids
→Polypeptide→Proteins
RNA:Single Strand
Adenine, Uracil,
Cytosine, Guanine
mRNA
t RNA
r RNA
DNA Transcription
1. Duplex DNA Seperation→
2 Single DNA Strands
Exposing the Nitrogenous
Base Sequences
Only One of the 2 DNA
Strands, Which Serves as
A Template,or Pattern(Gene), for the Synthesis of RNA.
2.Complementary
TTT TCT AAACGT
AAAAGATTT GCA
mRNAUUUUCUAAACGU
4x4x4=64
64 Possible 3-Letter Words
Translation
Protein Synthesis
20 Words in the Form
of 20 Common A.A.
Each Triplet Codes for
an Amino Acids
.
Table7.1 and 7.2
Ribosome:
Synthesis of Protein
Cellular Organelle that
Serves as the Meeting
Place for mRNA and
tRNA that carry A.A.
to Form Peptide
UUUUCUAAACGU
Phe--Ser--Lys—Arg
What is Gene Cloning?
Insert DNA Fragment to
Circle DNA Molecule-
-----Chimera
Recombinant DNA
PCR:Polymerase Chain
Reacyion
1995 Kary Mullis
1.Denaturation of DNA
94C
2.Annealing of the Primers (Oligonucleotides)
50---60C
3.Synthesis of New DNA 74C
4.Renaturation of DNA
Repeat the Cycle 25-30
Times
Gene Isolation by
Cloning
1. DNA Fragments
2. Construct Recombinant DNA
3. Introduce into Bacteria
4. Plate Out
5. Each Colony contains Multiple
Copies of just one
Recombinant DNA
Molecule
Gene Isolation by PCR
1. Polymerase Chain
Reaction
2.X Several Million
The Commercial Exploitation of Microorganism
1. Bacillus thuringiensis
Natural Insecticide
Thuringiensis Gene
選殖共生於玉米根部
可以防治玉米害蟲
2. Aureobasidium pullulans
Polysaccharides
Pullulan
3.Acinetobacter calcoaccticus
Polysaccharides
Emulsan
Biotransformation
Bioconversion
1. Steroid------Cortisone
37 steps
USD 200 dollars/g
11 steps
USD 6 dollars/g
2. Glucose—Sorbitol-----Sorbose-----Vitamin C
Glucose—Sorbitol
4 chemical steps
Biotransformation
1 chemical step
Examples:
1. Phenylpyruvic Acid
-----Phenylalanine
2. Cinnamic acid----Phenylalanine
Phe+Aspartic Acid---Aspartame
160 X Sweetness
Biotransformation
Growing Cell—休眠 Cell or Killed Cell
P.115
Enzymes:
1.Glucose---Fructose
Glucose Isomerase
Arthrobacter sp.
Bacillus sp.
2.Penicillin----6-APC
Penicillin Amidase
Escherichia coli
p.121
Through DNA Recombinant,the yield
Of Enzyme:20g/l
3. Chymosin
小牛胃中凝乳素
Milk---------Cheese
(with Flavor)
Protein:
1. Growth Hormone
Fish
Cow:secretion of milk
Human
2. Insulin
3. Interferon
4.Vaccine
GMP Requirement
p.123
Gene
Cell and Enzyme
Immobilization
Methods of Immobilization
1. Physical Adsorption
Physical Binding
Cell or Enzyme 以吸附
方式被固定裝置在管
柱中容易吸附也容易
脫離尤其在基質添加
時 Cell or Enzyme 容易
Autolyse 造成 Activity
喪失
2.Cross linking
以 Glutaraldehyde
Toluene diisocyanate
進行交聯形成不溶解
性間質
但 Cell or Enzyme
Activity 仍會流失
3.Entrapment Lattice
Collagen
Gelatin,Agar
Polyacrylamide
Cellulose
To form Polymer Matrix
Choice of Immobilization
Methods
Stability
Cost
Regenerability
Commercialization
Monoclonal Antibody
Polyclonal Antibody
Epitopes 抗原決定位
Chimeric
嵌人化
Humanized 擬人化
Total Human Monoclonal
Antibody
Making A Hybridoma
Antigen----Animal
Immunization
Blood Collection
Speen of Animal
RBC & Lymphocyte
Application of Monoclonal
Antibody
1. Diagnostic Kit
2. 增加敏感度與再現性
3. Therapy
Examples:
1. Hepatitis Virus
2. Polyoma
3. Adenovirus
Isolation & Purification of
MABs
Immunoaffinity
Chromatography
1. Specificity
2. Elution----Sharp Peak
3. Independent Target Protein
Immunotoxins as Therapeutic
Ricin----A,B Polypeptide
----S----S---A,B Chain Enter Cell Plasm
Ribosome---Dysfunction---
Cell’s Death
Immunotoxin:
1. MABs combine with A,B
Chain----Attack Cancer Cell
Stable in Human Body
2. MABs+Radioisotope
目標導向
放射線能殺死癌細胞
Radioisotope:
High Energy
Low Penetration
Short Half Life
Safety
Ex: At 13hrs
Y 64hrs
Human Gene Therapy
Definition
Somatic Cell Gene Therapy
Correction of Germ Cell
1. Hard To Isolation
2. Hurt Normal Cell