植物生物技術(shù)在生物制藥中的作用課件

(塊根/塊莖）儲藏器官特異啟動子GoldenPotatoβ-胡蘿卜素高含量馬鈴薯(塊根/塊莖）儲藏器官特異啟動子GoldenPotatoβ1功能基因（目的基因）：原始基因；改進版的基因，如單子葉植物密碼子偏好基因；雙子葉植物密碼子偏好基因；含信號肽基因（定位蛋白在細胞中表達的位置）等終止子：基因轉(zhuǎn)錄終止信號基因表達盒可以自己構(gòu)建，也可以公司合成功能基因（目的基因）：原始基因；改進版的基因，如單子葉植物密2第二步：轉(zhuǎn)基因及轉(zhuǎn)基因植株的培育農(nóng)桿菌法基因槍法第二步：轉(zhuǎn)基因及轉(zhuǎn)基因植株的培育農(nóng)桿菌法基因槍法3第三步：轉(zhuǎn)基因植株的鑒定、評價與篩選——轉(zhuǎn)基因植株鑒定：報告基因、PCR，SouthernBlot（DNA雜交）——轉(zhuǎn)基因植株評價（栽培與分析）：不同轉(zhuǎn)基因植株的表現(xiàn)不一樣——將符合要求的轉(zhuǎn)基因植株培育成新的品種第三步：轉(zhuǎn)基因植株的鑒定、評價與篩選——轉(zhuǎn)基因植株鑒定：報告4

對照番茄

轉(zhuǎn)Bt番茄Bt煙草蘇云金芽胞桿菌毒素蛋白基因（BT）對照煙草植物轉(zhuǎn)基因技術(shù)在農(nóng)作物大豆、油菜、玉米、棉花、番茄等植物上得到成功應(yīng)用對照番茄轉(zhuǎn)Bt番茄Bt煙草蘇云金芽胞桿菌毒素蛋白5將蘇云金桿菌殺蟲蛋白（Bt）基因轉(zhuǎn)入植物，培育出抗蟲新品種轉(zhuǎn)基因?qū)φ諏φ辙D(zhuǎn)基因抗蟲煙草抗蟲棉花將蘇云金桿菌殺蟲蛋白（Bt）基因轉(zhuǎn)入植物，培育出抗蟲新品種轉(zhuǎn)6抗蟲轉(zhuǎn)基因棉花獲大面積推廣抗蟲轉(zhuǎn)基因棉花獲大面積推廣7轉(zhuǎn)基因水稻抗性栽培試驗轉(zhuǎn)基因水稻抗性栽培試驗8IngoPotrykus:GoldenRice金色稻米

β-胡蘿卜素=維生素AScience（2000）IngoPotrykus:GoldenRice金色稻9Paineetal.Nat.Biotechnol.

23,482-487(2005)：優(yōu)化了基因，β-胡蘿卜素含量提高23倍Paineetal.Nat.Biotechnol.10轉(zhuǎn)基因植物生產(chǎn)藥物蛋白植物基因工程技術(shù)的發(fā)展為利用植物作為生物反應(yīng)器生產(chǎn)有用蛋白成為可能，誕生了分子農(nóng)業(yè)（分子農(nóng)場）=MolecularFarming利用基因工程技術(shù)將特定的基因轉(zhuǎn)入植物中，通過栽培轉(zhuǎn)基因植物生產(chǎn)具有治療、診斷、或者工業(yè)用途的蛋白或多肽。轉(zhuǎn)基因植物生產(chǎn)藥物蛋白11分子農(nóng)業(yè)示意圖分子農(nóng)業(yè)示意圖12藥物蛋白（人、家禽家獸用）：疫苗、抗體、生長因子、生長激素等?！⑸矬w系：產(chǎn)量高、成本低，但有時無活性——動物細胞體系：有活性；細胞培養(yǎng)環(huán)境和產(chǎn)品分離純化苛刻，成本高，純化難（病原、過敏原）藥物蛋白（人、家禽家獸用）：疫苗、抗體、生長因子、生長激素13——植物體系：可行：可以進行蛋白翻譯后的加工、形成有活性的物質(zhì)）；安全性好：人與植物沒有共患疾病，人對常用作物不過敏；成本低：農(nóng)作物種植投入少，可機械化大規(guī)模種植；可儲藏：1-3年以上使用方便：疫苗可以直接使用（香蕉、番茄等）——植物體系：14學(xué)術(shù)結(jié)構(gòu)和商業(yè)公司對分子農(nóng)業(yè)興趣濃厚，近20年發(fā)展迅速20多年來轉(zhuǎn)基因植物生產(chǎn)藥物蛋白的論文數(shù)量（近1000篇，100多項專利）學(xué)術(shù)結(jié)構(gòu)和商業(yè)公司對分子農(nóng)業(yè)興趣濃厚，近20年發(fā)展迅速20多15藥物蛋白的種類：疫苗、抗體、生長激素、細胞因子、膜蛋白、酶等藥物蛋白的種類：疫苗、抗體、生長激素、細胞因子、膜蛋白、酶等16美國2002-2005年批準進行試驗的植物來源的重組蛋白美國2002-2005年批準進行試驗的植物來源的重組蛋白17瀏覽以下網(wǎng)站可了解更多轉(zhuǎn)基因植物生產(chǎn)的藥物蛋白和工業(yè)用蛋白的研發(fā)情況：/PMPs-and-PMIPs.html瀏覽以下網(wǎng)站可了解更多轉(zhuǎn)基因植物生產(chǎn)的藥物蛋白和工業(yè)用蛋白的18TransgenicResearch13:245–259,2004

RecombinantproteinexpressionplasmidsoptimizedforindustrialE.colifermentationandplantsystemsproducebiologicallyactivehumaninsulin-likegrowthfactor-1intransgenicriceandtobaccoplants

MitraPanahi1,ZamanAlli1,XiongyingCheng1,LoubabaBelbaraka2,JaafarBelgoudi2,RavinderSardana1,JennyPhipps2&IllimarAltosaar1,*

1DepartmentofBiochemistry,MicrobiologyandImmunology,FacultyofMedicine,UniversityofOttawa,Canada2SteacieInstituteforMolecularSciences,NationalResearchCouncilofCanada,Ottawa,Ontario,K1AOR6,Canada

TransgenicResearch13:245–2519humaninsulin-likegrowthfactor-1=hIGF-1，人胰島素樣生長因子-1asingle-chainpeptideof70aminoacidresiduessharing50%homologywithhumaninsulin.SynthesisofhIGF-1:

Mainlyintheliverandregulatedbyseveralfactorssuchashumangrowthhormonesandinsulin.MainfunctionsofhIGF-1:

1Beingessentialfornormalfetalgrowthanddevelopment;2Stimulatingproliferationandsurvivalofmanycelltypes;3Promotingdifferentiationofalimitedcelltypes.

humaninsulin-likegrowthfact20LarondwarfismhIGF1playsanimportantroleinchildhoodgrowth.InabilitytomakeorrespondtohIGF1producesadistinctivetypeofgrowthfailuretermedLarondwarfism.hIGF1continuestohaveanaboliceffectsinadults.

LarondwarfismhIGF1playsani21TheclinicalimportanceofhIGF-1hIGF-1canincreaseinsulinsensitivityandpossesseshypoglycemiceffectssimilartoinsulin,sohIGF-1maybecomeasubstitutetherapeuticagentforthepeoplewhoseinsulinreceptorshavefunctionaldefects,especiallyforthosewhohaveoneormoreofthefollowingproblems:1.

Growthhormone-sensitive(Laron-type)syndrome;2.TypeAinsulinresistancesyndrome;3.Diabetes;4.Osteoporosis;5.Acquiredimmunodeficiencysyndrome(AIDS).

ThedemandforhIGF-1islikelytoincreaseinthefuture.TheclinicalimportanceofhIG22ProductionofhIGF-1RecombinanthIGF-1(rthIGF-1)hasbeenexpressedinseveraldifferenthost–vectorsystems:

Inyeast:8to55mg/Lculture(Vaietal,2000);

InE.coli:

8.5g/Lculture,butalmostalltheIGF-1werenotsoluble(inaggregates)

(Jolyetal.,1998);

Intransgenicrabbits:ahomozygousoffspringproduced678mg/Lmilk(Zinovievaetal.,1998).ProductionofhIGF-1Re23LimitationsinthepresentsystemsexpressinghIGF-1:1.Notsolubleinbacteria;2.Formationofnon-nativeproteinshavingdifferentbiologicalactivitiesinyeast;3.Transgeneinducedinstabilityofcertaincelllinesinmammaliancellcultures;4.Contaminationofanimal-basedproductswithhumanpathogensinmammaliancellcultures;5.Highcostsofmammaliancellcultures.Limitationsinthepresentsys24Potentialadvantagesofexpressingrecombinantproteinsinplants:1.Minimalriskofcontaminationbypathogenicmicroorganisms;2.Theabilitytoeffectpost-translationalmodificationsresultinginauthenticproducts;3.HighyieldsofstableproductsbyaddingplantsignalsequencesandincreasingG+Ccontentofthecodingsequences;4.Lowscale-upcosts;5.Edibletissuescanbetargetedforprecisetissue-specificaccumulationofrecombinantproteins.Potentialadvantagesofexpres25Figure1-1.TherthIGF-1expressionconstructsusedfortransformationoftobaccoandriceplants.ThegeneconstructscontainingthehIGF-1sequences(210bp)wereplacedunderthecontrolofthemaizeubiquitin1promoter(PanelsA–C).表達載體適合大腸桿菌的基因普通基因適合植物的基因Figure1-1.TherthIGF-1expre26ProductionoftobaccoandriceviaAgrobacterium-mediation

ThetransgenicplantsandhIGF-1geneexpressionintheplantswereconfirmedbySouthernblots,RT-PCRandWesternBlots.Productionoftobaccoandrice27DidthetransgenicplantsproducehIGF-1?

Yes.hIGF-1detectionbyELISAshowedthatallthetransgenictobaccoorriceplantsproducedhIGF-1intheirleaves,buttheyieldsvarieddramaticallyamongthem.Didthetransgenicplantsprod28Figure1-2.AmountsofrthIGF-1(ngofrthIGF-1/mgoftotalprotein)ineachplantextract.TobaccoRiceProductionofhIGF-1intransgenicplantsFigure1-2.AmountsofrthIGF-29PlantsGroupsPromoterSignalsequencesCodingsequenceRangeofhIGF1levelsMeansofhIGF1levelsTobaccopUIGF-1

ubiquitin1

NoSSE.coli-codonoptimized4–129ng/mg26±7ngTobaccopULamB-IGF-1

ubiquitin1BacterialLamBSSE.coli-codonoptimized5–73ng/mg22±4ngTobaccopUSYN-IGF-1

ubiquitin1RiceprolaminSSplant-codonoptimized8–241ng/mg70±13ngRice

pUIGF-1

ubiquitin1

NoSSE.coli-codonoptimized15–371ng/mg113±24ngPlantsGroupsPromoterSignalseq30(塊根/塊莖）儲藏器官特異啟動子GoldenPotatoβ-胡蘿卜素高含量馬鈴薯(塊根/塊莖）儲藏器官特異啟動子GoldenPotatoβ31功能基因（目的基因）：原始基因；改進版的基因，如單子葉植物密碼子偏好基因；雙子葉植物密碼子偏好基因；含信號肽基因（定位蛋白在細胞中表達的位置）等終止子：基因轉(zhuǎn)錄終止信號基因表達盒可以自己構(gòu)建，也可以公司合成功能基因（目的基因）：原始基因；改進版的基因，如單子葉植物密32第二步：轉(zhuǎn)基因及轉(zhuǎn)基因植株的培育農(nóng)桿菌法基因槍法第二步：轉(zhuǎn)基因及轉(zhuǎn)基因植株的培育農(nóng)桿菌法基因槍法33第三步：轉(zhuǎn)基因植株的鑒定、評價與篩選——轉(zhuǎn)基因植株鑒定：報告基因、PCR，SouthernBlot（DNA雜交）——轉(zhuǎn)基因植株評價（栽培與分析）：不同轉(zhuǎn)基因植株的表現(xiàn)不一樣——將符合要求的轉(zhuǎn)基因植株培育成新的品種第三步：轉(zhuǎn)基因植株的鑒定、評價與篩選——轉(zhuǎn)基因植株鑒定：報告34

對照番茄

轉(zhuǎn)Bt番茄Bt煙草蘇云金芽胞桿菌毒素蛋白基因（BT）對照煙草植物轉(zhuǎn)基因技術(shù)在農(nóng)作物大豆、油菜、玉米、棉花、番茄等植物上得到成功應(yīng)用對照番茄轉(zhuǎn)Bt番茄Bt煙草蘇云金芽胞桿菌毒素蛋白35將蘇云金桿菌殺蟲蛋白（Bt）基因轉(zhuǎn)入植物，培育出抗蟲新品種轉(zhuǎn)基因?qū)φ諏φ辙D(zhuǎn)基因抗蟲煙草抗蟲棉花將蘇云金桿菌殺蟲蛋白（Bt）基因轉(zhuǎn)入植物，培育出抗蟲新品種轉(zhuǎn)36抗蟲轉(zhuǎn)基因棉花獲大面積推廣抗蟲轉(zhuǎn)基因棉花獲大面積推廣37轉(zhuǎn)基因水稻抗性栽培試驗轉(zhuǎn)基因水稻抗性栽培試驗38IngoPotrykus:GoldenRice金色稻米

β-胡蘿卜素=維生素AScience（2000）IngoPotrykus:GoldenRice金色稻39Paineetal.Nat.Biotechnol.

23,482-487(2005)：優(yōu)化了基因，β-胡蘿卜素含量提高23倍Paineetal.Nat.Biotechnol.40轉(zhuǎn)基因植物生產(chǎn)藥物蛋白植物基因工程技術(shù)的發(fā)展為利用植物作為生物反應(yīng)器生產(chǎn)有用蛋白成為可能，誕生了分子農(nóng)業(yè)（分子農(nóng)場）=MolecularFarming利用基因工程技術(shù)將特定的基因轉(zhuǎn)入植物中，通過栽培轉(zhuǎn)基因植物生產(chǎn)具有治療、診斷、或者工業(yè)用途的蛋白或多肽。轉(zhuǎn)基因植物生產(chǎn)藥物蛋白41分子農(nóng)業(yè)示意圖分子農(nóng)業(yè)示意圖42藥物蛋白（人、家禽家獸用）：疫苗、抗體、生長因子、生長激素等?！⑸矬w系：產(chǎn)量高、成本低，但有時無活性——動物細胞體系：有活性；細胞培養(yǎng)環(huán)境和產(chǎn)品分離純化苛刻，成本高，純化難（病原、過敏原）藥物蛋白（人、家禽家獸用）：疫苗、抗體、生長因子、生長激素43——植物體系：可行：可以進行蛋白翻譯后的加工、形成有活性的物質(zhì)）；安全性好：人與植物沒有共患疾病，人對常用作物不過敏；成本低：農(nóng)作物種植投入少，可機械化大規(guī)模種植；可儲藏：1-3年以上使用方便：疫苗可以直接使用（香蕉、番茄等）——植物體系：44學(xué)術(shù)結(jié)構(gòu)和商業(yè)公司對分子農(nóng)業(yè)興趣濃厚，近20年發(fā)展迅速20多年來轉(zhuǎn)基因植物生產(chǎn)藥物蛋白的論文數(shù)量（近1000篇，100多項專利）學(xué)術(shù)結(jié)構(gòu)和商業(yè)公司對分子農(nóng)業(yè)興趣濃厚，近20年發(fā)展迅速20多45藥物蛋白的種類：疫苗、抗體、生長激素、細胞因子、膜蛋白、酶等藥物蛋白的種類：疫苗、抗體、生長激素、細胞因子、膜蛋白、酶等46美國2002-2005年批準進行試驗的植物來源的重組蛋白美國2002-2005年批準進行試驗的植物來源的重組蛋白47瀏覽以下網(wǎng)站可了解更多轉(zhuǎn)基因植物生產(chǎn)的藥物蛋白和工業(yè)用蛋白的研發(fā)情況：/PMPs-and-PMIPs.html瀏覽以下網(wǎng)站可了解更多轉(zhuǎn)基因植物生產(chǎn)的藥物蛋白和工業(yè)用蛋白的48TransgenicResearch13:245–259,2004

RecombinantproteinexpressionplasmidsoptimizedforindustrialE.colifermentationandplantsystemsproducebiologicallyactivehumaninsulin-likegrowthfactor-1intransgenicriceandtobaccoplants

MitraPanahi1,ZamanAlli1,XiongyingCheng1,LoubabaBelbaraka2,JaafarBelgoudi2,RavinderSardana1,JennyPhipps2&IllimarAltosaar1,*

1DepartmentofBiochemistry,MicrobiologyandImmunology,FacultyofMedicine,UniversityofOttawa,Canada2SteacieInstituteforMolecularSciences,NationalResearchCouncilofCanada,Ottawa,Ontario,K1AOR6,Canada

TransgenicResearch13:245–2549humaninsulin-likegrowthfactor-1=hIGF-1，人胰島素樣生長因子-1asingle-chainpeptideof70aminoacidresiduessharing50%homologywithhumaninsulin.SynthesisofhIGF-1:

Mainlyintheliverandregulatedbyseveralfactorssuchashumangrowthhormonesandinsulin.MainfunctionsofhIGF-1:

1Beingessentialfornormalfetalgrowthanddevelopment;2Stimulatingproliferationandsurvivalofmanycelltypes;3Promotingdifferentiationofalimitedcelltypes.

humaninsulin-likegrowthfact50LarondwarfismhIGF1playsanimportantroleinchildhoodgrowth.InabilitytomakeorrespondtohIGF1producesadistinctivetypeofgrowthfailuretermedLarondwarfism.hIGF1continuestohaveanaboliceffectsinadults.

LarondwarfismhIGF1playsani51TheclinicalimportanceofhIGF-1hIGF-1canincreaseinsulinsensitivityandpossesseshypoglycemiceffectssimilartoinsulin,sohIGF-1maybecomeasubstitutetherapeuticagentforthepeoplewhoseinsulinreceptorshavefunctionaldefects,especiallyforthosewhohaveoneormoreofthefollowingproblems:1.

Growthhormone-sensitive(Laron-type)syndrome;2.TypeAinsulinresistancesyndrome;3.Diabetes;4.Osteoporosis;5.Acquiredimmunodeficiencysyndrome(AIDS).

ThedemandforhIGF-1islikelytoincreaseinthefuture.TheclinicalimportanceofhIG52ProductionofhIGF-1RecombinanthIGF-1(rthIGF-1)hasbeenexpressedinseveraldifferenthost–vectorsystems:

Inyeast:8to55mg/Lculture(Vaietal,2000);

InE.coli:

8.5g/Lculture,butalmostalltheIGF-1werenotsoluble(inaggregates)

(Jolyetal.,1998);

Intransgenicrabbits:ahomozygousoffspringproduced678mg/Lmilk(Zinovievaetal.,1998).ProductionofhIGF-1Re53LimitationsinthepresentsystemsexpressinghIGF-1:1.Notsolubleinbacteria;2.Formationofnon-nativeproteinshavingdifferentbiologicalactivitiesinyeast;3.Transgeneinducedinstabilityofcertaincelllinesinmammaliancellcultures;4.Contaminationofanimal-basedproductswithhumanpathogensinmammaliancellcultures;5.Highcostsofmammaliancellcultures.Limitationsinthepresentsys54Potentialadvantagesofexpressingrecombinantproteinsinplants:1.Minimalriskofcontaminationbypathogenicmicroorganisms;2.Theabilitytoeffectpost-translationalmodificationsresultinginauthenticproducts;3.HighyieldsofstableproductsbyaddingplantsignalsequencesandincreasingG+Ccontentofthecodingsequences;4.Lowscale-upcosts;5.Edibletissuescanbetargetedforprecisetissue-specificaccumulationofrecombinantproteins.Potentialadvantagesofexpres55Figure1-1.TherthIGF-1expressionconstructsusedfortransformationoftobaccoandriceplants.ThegeneconstructscontainingthehIG