质粒构建课件

质粒构建11孟庆明2019-04-09主要内容质粒的概念及特点质粒构建的基本步骤和原理一、质粒的概念载体（vector;carrier;vehicle）可以插入核酸片段、能携带外源核酸进入宿主细胞，并在其中进行独立和稳定的自我复制的核酸分子。基因工程中广泛应用的载体多来自人工改造的细菌质粒、噬菌体或病毒核酸等。多数载体是DNA分子，但某些RNA分子也能用做载体。质粒（plasmid）独立于染色体外能够进行自主复制的遗传单位，包括真核生物的细胞器和细菌中染色体以外的双链、闭环DNA分子。现在习惯上用来专指细菌、酵母菌和放线菌等生物中染色体以外的DNA分子。在基因工程中质粒常被用做基因的载体。分为紧密控制型(Stringentcontrol)和松驰控制型(Relaxedcontrol)4①在宿主细胞中能保存下来并能大量复制，不影响受体细胞正常生命活动；②有多个限制酶切点（MCS），而且每种酶的切点最好只有一个，可适于多种限制酶切割的DNA插入；③含有复制起始位点，能够独立复制；④有一定的标记基因，便于进行筛选。如pEGFP-n1质粒携带卡那霉素抗性基因，pcs2质粒携带氨苄青霉素抗性基因；⑤载体DNA分子大小应合适，分子量小（1-10KD）、多拷贝、松驰控制型，便于操作及大量扩增，如我们实验室常用的pcDNA3.1,pcs2-6myc,pEGFP-n1等。二、质粒的特点55三、质粒图谱阅读第一步：首先看Ori的位置，了解质粒的类型（原核/真核/穿梭质粒）。第二步：再看筛选标记，如抗性，决定使用什么筛选标记。Ampr氨苄抗性；kanr卡那霉素抗性。neor使G418失活。第三步：看多克隆位点（MCS）。是否具有多个限制酶的单一切点。第四步：再看外源DNA插入片段大小。质粒一般只能容纳小于10Kb的外源DNA片段。第五步：是否含有表达系统元件，即启动子－核糖体结合位点－克隆位点－转录终止信号。这是用来区别克隆载体与表达载体。五步阅读质粒图谱四、质粒构建的基本步骤和原理背景介绍：重组DNA技术（recombinantDNAtechnology）是基因功能研究的基本方法。应用该技术可以对DNA分子进行剪切和重新连接，构成重组DNA分子，然后把它导入宿主细胞，进而扩增相关DNA片段，表达相关基因的产物。克隆（clone）是指由一个细胞经过无性繁殖以后形成的子代群体。构建DNA重组体并导入宿主细胞建立无性繁殖体系，即DNA的分子克隆（molecularcloning）过程。因此，重组DNA技术又称为基因克隆技术或基因工程技术。基因克隆Genecloning（以hBex2为例）Targetgene:hBex2VectorselectionPrimerdesignAmplification,digestion&ligationTransformation&IdentificationTargetgeneTargetgeneTargetgeneTargetgeneGenecloningTargetgene:hBex2VectorselectionPrimerdesignAmplification,digestion&ligationTransformation&IdentificationVectorswithfluorescenttag:pEGFPC1;pEGFPN1;pEYFP;pSUPER;pCS2(GFP)……Vectorswithnon-fluorescenttag:pKH3(3HA);pCS2(6myc);pcDNA3.1(flag);pCMV5(flag);pGEX(GST);……VECTORSgenome-stanford.edu/vectordb/GenecloningTargetgene:hBex2VectorselectionPrimerdesignAmplification,digestion&ligationTransformation&IdentificationPrimerdesignPrimerdesignSense:5’ATGGAGTCCAAAGAGGAACG3’PrimerdesignAntisense5’TCAGGGCATAAGGCAAAACTCATCG3’PrimerdesignSense:5’ATGGAGTCCAAAGAGGAACG3’(Forword)Antisense5’TCAGGGCATAAGGCAAAACTCATCG3’(Reverse)PrimerdesignBeforeorderit…Thinkaboutwhatshouldbeincludedinlinker:RestrictionsiteselectionProtectiveoligonucleotidesforeffectivecleavageKozaktranslationinitiationsite(GCCGCCA/GCCATGG)Openreadingframes(forfusionprotein)Stopcodon?InsertS?REREORF?K?SearchforrestrictionsitesSearchforrestrictionsitesProtectiveoligonucleotidesforeffectivecleavageCleavageClosetotheEndofDNAFragments(oligonucleotides)CGGAATTCCGGGATCCEcoRIBamHIneb/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.aspKozaktranslationinitiationsiteInitiationoftranslationineukaryotes.Consensussequence:GCCGCCA/GCCATGG-3,1,2,3,4OpenreadingframesXX5’GAATTCEcoRIBamHIGGATCC3’……EGFPInsert(ATGXXX……XXX)Product(fusionprotein):Targetprotein+GFPEcoRI:GAATTCTGHindIII:XXXOpenreadingframesStopcodonTAGTGATAANOAAntisenselinker:AAGCTTBamHI:GGATCCAHindIII:AAGCTTEcoRI:GAATTCBamHI:GGATCCXXXSenselinker:OpenreadingframesPrimerdesign:StopcodonAntisense5’GGGCATAAGGCAAAACTCATCG3’Antisense5’TCAGGGCATAAGGCAAAACTCATCG3’Sense：hBex2/EcoRI5’-CGGAATTCATGGAGTCCAAAGAGGAACG-3’Antisense：hBex2/BamHIAntisense:5’-CGGGATCCCGGGGCATAAGGCAAAACTCATCG-3’Sense:5’-CGGAATTCATGGAGTCCAAAGAGGAACG-3’5’-CGGGATCCCGGGGCATAAGGCAAAACTCATCG-3’GenecloningTargetgene:hBex2VectorselectionPrimerdesignAmplification,digestion&ligationTransformation&IdentificationAmplification(PCR):94°C3’94°C45’’Tm45’’30-40cycles72°C1’72°C10’Template(cDNAorPlasmid)SenseprimerAntisenseprimerDNAPolymerase(pfu)dNTPmixBufferddH2ODigestion&ligationPCRproductorvectorEnzyme1Enzyme210XBuffer100XBSA(?)ddH2O37°C2~3hPCRproductVectorT4DNAligase10XligasebufferddH2ORT1~2hor16°CO/NCTTCGGATCCACCGCGGTGAATTCGAAGEcoRIBamHIGFPEcoRIBamHI5’GATCCGC5’AATTCGCG3’insertG3’5’CGGAATTCEcoRIBamHIGCGGATCC3’insertGAATTC3’5’CGGGATCCGCEcoRI/BamHIdigestionCGGTGGATCCCGXXXXXCAGAATTCGAAGCTTCGAATTCTGXXXXXCGGGATCCACCGGFPEcoRIBamHIEcoRI/BamHIdigestionCTTCGGATCCACCGCGGTGAATTCGAAGinsertGT4DNALigaseGFPGCGGATCCGCAATTCDigestion&ligationGenecloningTargetgene:hBex2VectorselectionPrimerdesignAmplification,digestion&ligationTransformation&IdentificationTransformation&IdentificationPCRidentification:TaqDNApolymeraseDigestionidentificationSequencingidentification:sangongorinvitrogen