Introduction to

Biological Science生物科技概論

By: Chung W.Wu,M.S.,R.Ph Ext:---5213

Ancient Biological Science1. Food Preparation Beer,Cheese2. Fermentation Microbial Food--------------Perfume3. Alcohol Fermentation Distillation Alcohol Concentration

1st World War Germany Glucose---------Glycerol British Sugar---------------------Acetone+Butanol Clostridium acetobutylicum Carbon Dioxide Hydrogen Gas:Explosive

2nd World War1940 FlemingPenicillium notatum Penicillin:Broad Spectrum Antibiotic 2mg/lPenicillium chrysogenum UV,X-Ray—Mutation 20mg/lStreptomyces griseus Stretomycin

Genetic EngineeringDNA Recombination RecombinantDeoxyribonucleic AcidChemical Composition1. Phosphate2. Pentose3. Nitrogenous Base Adenine Thymine Cytosine Guanine

Principles of DNA RecombinantDNA 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 Site4. Sticky Ends

Expression of Cloned Genes1. Transcription2. Translation3. Post-Translation

The Molecular Basis of Heredity: DNA & RNA

Nuclear ComponentsDNA→Transcription→RNA→Translation→Amino Acids→Polypeptide→Proteins RNA:Single Strand Adenine, Uracil, Cytosine, Guanine mRNA t RNA r RNA

DNA Transcription1. 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.ComplementaryTTT TCT AAACGTAAAAGATTT GCAmRNAUUUUCUAAACGU4x4x4=6464 Possible 3-Letter Words

TranslationProtein Synthesis20 Words in the Formof 20 Common A.A.Each Triplet Codes foran Amino Acids

Ribosome:Synthesis of ProteinCellular Organelle thatServes as the Meeting Place for mRNA andtRNA that carry A.A.to Form Peptide

UUUUCUAAACGUPhe--Ser--Lys—Arg

What is Gene Cloning?Insert DNA Fragment toCircle DNA Molecule------Chimera Recombinant DNA

PCR:Polymerase Chain Reaction1995 Kary Mullis1.Denaturation of DNA 94C2.Annealing of the Primers (Oligonucleotides) 50---60C 3.Synthesis of New DNA 74C4.Renaturation of DNARepeat the Cycle 25-30 Times

Gene Isolation by Cloning1. DNA Fragments2. Construct Recombinant DNA3. Introduce into Bacteria4. Plate Out5. Each Colony contains Multiple

Copies of just one

Gene Isolation by PCR1. Polymerase Chain Reaction2. 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

BiotransformationGrowing Cell— 休眠 Cell or Killed Cell

Enzymes: 1.Glucose---Fructose Glucose Isomerase Arthrobacter sp. Bacillus sp.

2.Penicillin----6-APC Penicillin Amidase Escherichia coli 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. Insulin3. Interferon4. VaccineGMP Requirement

Cell and Enzyme ImmobilizationMethods of Immobilization1. 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 CelluloseTo 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 AntibodyMaking A HybridomaAntigen----AnimalImmunizationBlood Collection Speen of Animal RBC & Lymphocyte

Application of Monoclonal Antibody1. Diagnostic Kit2. 增加敏感度與再現性3. Therapy

Examples:1. Hepatitis Virus2. Polyoma3. Adenovirus

Isolation & Purification of MABsImmunoaffinity Chromatography1. Specificity2. Elution----Sharp Peak3. Independent Target Protein

Immunotoxins as Therapeutic Ricin----A,B Polypeptide ----S----S----A,B Chain Enter Cell PlasmRibosome---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 TherapySomatic Cell Gene TherapyCorrection of Germ Cell 1. Hard To Isolation 2. Hurt Normal Cell