IntroductiontoBiologicalScience生物科技概論By:ChungW.Wu,M.S.,R.PhExt:---52131AncientBiologicalScience1.FoodPreparationBeer,Cheese2.FermentationMicrobialFood--------------Perfume3.AlcoholFermentationDistillationAlcoholConcentration21stWorldWarGermanyGlucose---------GlycerolBritishSugar---------------------Acetone+ButanolClostridiumacetobutylicumCarbonDioxideHydrogenGas:Explosive32ndWorldWar1940FlemingPenicilliumnotatumPenicillin:BroadSpectrumAntibiotic2mg/lPenicilliumchrysogenumUV,X-Ray—Mutation20mg/lStreptomycesgriseusStretomycin4GeneticEngineeringDNARecombinationRecombinantDeoxyribonucleicAcidChemicalComposition1.Phosphate2.Pentose3.NitrogenousBaseAdenineThymineCytosineGuanine5PrinciplesofDNARecombinantDNASectioncan’tReplicatebyitself.1.CloningVector

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.RestrictionEndonuclease(DNase)RecognitionSite4.StickyEnds6ExpressionofClonedGenes1.Transcription2.Translation3.Post-Translation

7TheMolecularBasisofHeredity:DNA&RNA

NuclearComponentsDNA→Transcription→RNA→Translation→AminoAcids→Polypeptide→ProteinsRNA:SingleStrandAdenine,Uracil,Cytosine,GuaninemRNAtRNArRNA8

DNATranscription1.DuplexDNASeperation→2SingleDNAStrandsExposingtheNitrogenousBaseSequencesOnlyOneofthe2DNAStrands,WhichServesasATemplate,orPattern(Gene),fortheSynthesisofRNA.9

2.ComplementaryTTTTCTAAACGTAAAAGATTTGCAmRNAUUUUCUAAACGU4x4x4=6464Possible3-LetterWords10TranslationProteinSynthesis20WordsintheFormof20CommonA.A.EachTripletCodesforanAminoAcids11Ribosome:SynthesisofProteinCellularOrganellethatServesastheMeetingPlaceformRNAandtRNAthatcarryA.A.toFormPeptideUUUUCUAAACGUPhe--Ser--Lys—Arg12WhatisGeneCloning?InsertDNAFragmenttoCircleDNAMolecule------ChimeraRecombinantDNA13PCR:PolymeraseChainReaction1995KaryMullis1.DenaturationofDNA94C2.AnnealingofthePrimers(Oligonucleotides)50---60C3.SynthesisofNewDNA74C4.RenaturationofDNARepeattheCycle25-30Times14GeneIsolationbyCloning1.DNAFragments2.ConstructRecombinantDNA3.IntroduceintoBacteria4.PlateOut5.EachColonycontainsMultipleCopiesofjustone15GeneIsolationbyPCR1.PolymeraseChainReaction2.XSeveralMillion16TheCommercialExploitationofMicroorganism1.BacillusthuringiensisNaturalInsecticideThuringiensisGene

選殖共生於玉米根部可以防治玉米害蟲172.AureobasidiumpullulansPolysaccharidesPullulan183.AcinetobactercalcoaccticusPolysaccharidesEmulsan19BiotransformationBioconversion1.Steroid------Cortisone37stepsUSD200dollars/g11stepsUSD6dollars/g20Glucose—Sorbitol-----Sorbose-----VitaminCGlucose—Sorbitol4chemicalstepsBiotransformation1chemicalstep21Examples:1.PhenylpyruvicAcid-----Phenylalanine2.Cinnamicacid-----PhenylalaninePhe+AsparticAcid----Aspartame160XSweetness22BiotransformationGrowingCell—休眠CellorKilledCell23Enzymes:1.Glucose---FructoseGlucoseIsomeraseArthrobactersp.Bacillussp.242.Penicillin----6-APCPenicillinAmidaseEscherichiacoliThroughDNARecombinant,theyieldOfEnzyme:20g/l253.Chymosin

小牛胃中凝乳素Milk---------Cheese(withFlavor)26Protein:1.GrowthHormoneFishCow:secretionofmilkHuman272.Insulin3.Interferon4.VaccineGMPRequirement28CellandEnzymeImmobilizationMethodsofImmobilization1.PhysicalAdsorptionPhysicalBindingCellorEnzyme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CellorEnzyme容易Autolyse造成Activity喪失292.Crosslinking

以GlutaraldehydeToluenediisocyanate

進行交聯形成不溶解性間質但CellorEnzymeActivity仍會流失303.EntrapmentLatticeCollagenGelatin,AgarPolyacrylamideCelluloseToformPolymerMatrix31ChoiceofImmobilizationMethodsStabilityCostRegenerabilityCommercialization32MonoclonalAntibodyPolyclonalAntibodyEpitopes抗原決定位Chimeric嵌人化Humanized擬人化33TotalHumanMonoclonalAntibodyMakingAHybridomaAntigen----AnimalImmunizationBloodCollectionSpeenofAnimalRBC&Lymphocyte34ApplicationofMonoclonalAntibody1.DiagnosticKit2.增加敏感度與再現性3.Therapy35Examples:1.HepatitisVirus2.Polyoma3.Adenovirus36Isolation&PurificationofMABsImmunoaffinityChromatography1.Specificity2.Elution----SharpPeak3.IndependentTargetProtein37ImmunotoxinsasTherapeuticRicin----A,BPolypeptide----S----S----A,BChainEnterCellPlasmRibosome---Dysfunction---Cell’sDeath38Immunotoxin:MABscombinewithA,BChain----AttackCancerCellStableinHumanBody392.MABs+Radioiso