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§4 基因克隆的载体系统 PowerPoint PPT Presentation


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§4 基因克隆的载体系统. 4.1 质粒( plasmid )载体 大肠杆菌主要的天然质粒类型: ① F 质粒:致育因子或性因子 ② R 质粒:抗药性因子 ③ Col 质粒:产生大肠杆菌素因子 基因工程中使用的质粒载体都是在天然质粒的基础上人工改造而成的。质粒载体是重组 DNA 研究中最常用的也是最重要的基因克隆载体。. 4.1.1 质粒的生物学特性 4.1.1.1 质粒 DNA 及其构型 绝大多数质粒都是环状双链 DNA 分子 质粒 DNA 的构型:

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§4 基因克隆的载体系统

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4

4

  • 4.1 plasmid

  • F

    R

    Col

  • DNA


4

  • 4.1.1

  • 4.1.1.1 DNA

  • DNA

  • DNA

  • SC cccDNA

  • OC DNAocDNA

  • L

    DNA


4

2F

  • 4.1.1.2 DNA

  • 1


4

  • 4.1.1.3 DNA

  • ColE1

  • 2

    bomColE1 DNA

    Mob

    mob

    ColE1

    bom


4

  • 4.1.1.4 DNA

  • 1DNA2DNA

  • 1~3

    10~60


4

  • 4.1.1.5


4


4

  • 4.1.2 DNA

  • DNASDSDNADNADNARNA

  • 4.1.2.1

  • pH12.0~12.5DNAcccDNADNADNARNAcccDNA


4

  • 4.1.2.2

    DNADNA

  • 4.1.2.3 CsCl

    DNADNA -


4

  • 4.1.2.4 DNA

  • 1

  • 2

  • 3


4

  • 4.1.3

  • 4.1.3.1

  • 4.1.3.2

    1

    22

    3

    4


4

  • 4.1.3.3

  • 1


4

  • 2

  • DNAtetrDNA

  • TetrTets


4

  • tetrTets Tets


4

  • 4.1.3.4

  • DNACol1Pmb1

  • Col1DNADNA10~12h1000~3000DNA


4

  • DNA

  • DNADNADNA


4


4

  • pKN80

  • Pkn80MukilpKN80HpaIHindIIIDNA

    AmprMu

    HpaIHindIII

    DNA


4


4

  • 4.1.4

  • 4.1.4.1 pSC101

  • pSC101

  • 1~29.09Kbtetr

  • EcoRI7HindIIIBamHISalI3DNAtetr


4

  • pSC1011

  • Ampr pSC101


4

  • pSC1012

  • pSC101

  • rDNA


4

  • 4.1.4.2 pBR322

  • pBR322 3pSF2124Tn3amprpSC101tetrCoLEIpMB1DNA


4

  • pBR322

  • 4363bpDNA6kbDNA

  • amprtetr

  • 24

    BamHI9tetramprPstI3DNA


4

  • pBR322 tetr


4

  • 4.1.4.3 pUC

  • pUCpBR322MCS5-MCSlacZ

  • pUC4

    pBR322

    ampr

    -lacZ

    -DNAlacZ

    lacZ5-MCS

    lacZ


4

pUC18 pUC19 MCS


4

  • pUC

  • 2.7kb

    500~700/

  • lacZ-X-gal

  • MCSM13mp8MCS

    MCSDNA

    M13mp8

    DNA

    pUC


4

  • 4.1.4.4

  • 1pBR327

    pBR327pBR322pBR3221.09kbDNA

  • pBR3272

  • 30~

    45

  • bom


4

  • 2pGEM-3Z

  • pGEM-3ZpUCpUC lacZEcoRI13MCS

  • pGEM-3ZpUC2T7SP6MCST7SP6RNA

  • pGEM-3Z pGEM-3ZT7SP6

  • pSP64Psp65pUC


4

  • pGEM-3Z

    pGEM-3Z


4

pSP64Psp65SP6

MCS


4

  • 3

  • --DNA

  • pBPV-BV1-


4

  • -

  • aAmps

    bLEU

    c

    d

    Ampr

    e

    leu


4

  • 4.2

  • 4.2.1

  • 31106dalDNA48.502kb 61 DNA DNA12 5 DNAcos


4

  • 4.2.1.1

  • 1

  • DNA

    DNA


4

  • 2

  • A

    DNADNA10kbcDNADNA

  • DNADNA

  • - lac5lacZlac5DNA-lacIPTGXgalDNAlacIPTGXgal


4

  • B

  • DNADNADNADNADNA15kbDNA


4

  • 3Charen

  • Charencharen bacteriophage Charen -lac5lac

  • Charen

  • CharenDNA

  • DNADNA


4

  • 4.2.1.2 DNA

  • 1 DNA

  • transfection:DNA

  • 103~104DNA

  • 2 DNA

  • DNA


4

  • 3 DNA

  • DNA DNA5% DNA75% DNA75%~105%

  • DNA48.502kb28kb51kb DNA23kb 15kb


4

  • 4.2.1.3

  • 1 cI

    cI

  • 2lacZ

  • 3sPi

    redgamP22P2


4

  • 4.2.2

  • 4.2.2.1

  • cosmidcos site-carrying plasmidcos cos

  • DNA

  • pHC79

  • cro-rIIBgl IIpBR322DNA1459~1666bpSau3ABgl II

  • Bgl IIcos1.78kb DNABgl IIpHC79


4

pHC79cos

DNA

DNA

ampr

tetr

pHC79

pBR322

31~45kb


4

  • 4.2.2.2

  • 1 DNA cos

  • 2

  • 3cos5~7kb45kbDNA


4

  • 4


4

  • 4.2.2.3

    cosmid cloning:DNA

    DNADNADNAcos40kbDNA

    costerminaseTercos

    DNA-coscos


4


4

  • 4.2.3

  • M13f1 fd 6.4kbDNA

  • DNA

    DNAreplication form DNARF DNA

    DNADNA

    RF DNAssDNA

    DNADNA

    M13 6DNA

    DNADNA

    DNADNA


4

  • 4.2.3.1 M13 M13

  • 1M13

    M13

    ,

    900nm9nm

    +

    DNA


4

M13 F

M13

DNA

M13 F

III

ssDNA

F

M13ssDNAII

RF DNADNA

M13

M13 DNA

M13 DNA


4

M13

M13

a+DNA

RF DNA

bRF DNA

cRF DNAM13mRNA

dM13IIRF DNA

M13

eDNAI

fM13II

g


4

  • M13 RF DNA200VDNARF DNADNA

  • VDNA-VDNAM13 M13

  • M13 1/2~3/41000

  • RF DNAM13 DNA


4

  • 2M13

  • -

  • M13--XgelX-gel

    -Xgel5--4--3---D-5--4--

  • DNA-M13--


4

  • laclacZM15M1511~41-M15-2~92-M15--

  • -HindII-M13lacHindIIM13 FM15

  • M13 lacHindIIlac -200RF DNAlac+10


4

  • lacIqM13 RF DNAlac Iq lacIPTG--D-lacHindII

  • IPTG

  • -

  • -

  • IPTG

  • M13JM101lacZFM15lac Iq laclacIPTG

IPTG


4

lacHindII=lacZOPI

lacIlacP

lacOlacZ.

lacZ:145

NMU-N-

  • M13

  • M13

    IIIV

    507

    54986005

    M13 DN

    A

    DNA

    M13

    M13 mpn

    nM13mp8

    M13mp9

M13


4

  • lac HindII

  • BsuIM13 RF DNA10lac HindIIDNA lac HindIIBsuIlac HindIIDNA

  • lac HindIIAvaIIBgl IIPvuIPvuIIEcoRIHindIII1978B.GronenbornJ.MessingEcoRIM13mp1lac HindIIM13mp2


4

M13mp1

M13mp2

M13mp15

GGATT

C2

G

EcoRI


4

M13mp2M13mp7


4

M13


4

  • M13

  • lac-HindII

  • M13DNADNADNA


4

DNA

M13


4

M13

M13mp10/M13mp11


4

  • 3phage display vectors

  • M13DNA3~5III

  • G.P.Smith1985phage display

  • DNAIIIIII


4


4

  • hGH

    ahGH cDNA

    b

    chGH

    dhGH-

    e

    f


4

  • 4.2.4

  • 4.2.4.1

    M13DNA

    1DNA

    2DNA

    3DNA1.5kb

    phagemid phasmid


4

  • 4.2.4.2

    1M133kb

    10kbDNA

    2

    DNADNA

    II

    M13DNA

    3DNA


4

  • 4.2.4.3 pUC118pUC119

    pUC118pUC119pUC18pUC19M13IG

    IG476bp

    M13

    pUC18

    pUC19NdeI


4

pUC118pUC119

pUC118

pUC119


4

pUC118

pUC119


4

M13K07 8.7kb

  • DNA

    M13

    M13K07

    M13IG

    AvaI

    M13m

    pI

    p15A

    Tn903

pUC118 pUC119

M13K07


4

  • pUC118pUC119

    1cccDNA10kbDNA

    2ampr

    3500

    DNA

    4DNA

    lacZ5-

    Xgal-IPTG

    5

    6pUC118pUC119MCS

    DNADNA

    7DNA


4

  • pUC118/pUC119

    1DNA

    2lacZ15

    Amps

    3Xgal-

    IPTG


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