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第十章 外源基因的表达 PowerPoint PPT Presentation


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第十章 外源基因的表达. 基因重组的 主要目的 是要使目的基因在某一细胞中能得到高效表达,即产生人们所需要的高产目的的基因产物,如蛋白质、多肽类生物药物。. 基因工程技术的 核心 是 基因表达技术 。 基因表达 是指结构基因在调控序列的作用下转录成 mRNA ,经加工后在核糖体的协助下又转译出相应的基因产物 —— 蛋白质,再在受体细胞环境中经修饰而显示出相应的功能。从基因到有功能的产物这整个转录、转译及所有的加工过程就是基因表达的过程,它是在一系列酶和调控序列的共同作用下完成的。. 基因表达在原核生物与真核生物中的差别?.

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第十章 外源基因的表达

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4607739


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mRNA


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  • RNAmRNA mRNAmRNA

  • hnRNA53mRNAmRNA


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1

1.1

lactrpPR PL tac

T7


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1.2 mRNA

mRNA

mRNA

mRNA


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1.3 mRNA

mRNA

  • AUG(ATG)

  • SD16S rRNAAGGAGG4

  • SD39

  • mRNA


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major codon

rare codon

mRNA


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1.4


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1.5

DNA

RNA


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2

2.1

promoterRNADNARNA1035TATA

*


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2.1.1

  • CATA/G

  • Pribnow 10bpTATA10

  • Sextama35bpTTGACA35


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3

5

-35

-10

TTGACA

16-19 bp

TATAAT

5-9 bp

T82T84G78A65C54A45

T80A95T45A60A50T96

Transcriptional start site

The prokaryotic promoter

The sequence of DNA needed for RNA polymerase to bind to the template and accomplish the initiation reaction.


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2.1.1

rRNA

mRNA

tRNA


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rRNArRNA

UCE

180190bp


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TATAHognessATDNA2030bp

CAAT75bpRNA

GC100300bp


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tRNA5SRNA

U6RNA7SKRNA


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2.1.3

RNA

RNA

(2)( 2 )


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sfactor:


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2.1.4

PCR


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2.2

enhancerDNA


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2.3

RNA


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()RNARNA

U


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5090bp GCRNAURNA

2.0105NTPNTP RNA


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RNARNAATP53RNARNA3OHRNARNA


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2.4

attenuatormRNA

trp

trp412341-23-42-3

tRNATrptrptrptRNATrptrp41trp 2-33-4trptrptrp422-33-4RNA


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Low Trp

High Trp


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2.5

  • insulator


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2.6

RNAantisence RNAmRNARNARNADNAmRNARNAantisence techology

RNADNA

DNA


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  • RNARNARNADNADNA

  • RNADNA

  • RNAmRNA5

  • mRNApoly(A)mRNA

  • mRNA


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  • mRNA5 SDmRNA

  • mRNA5

SDSD sequenceShine-DalgarnomRNAAUGAGGAGGG16S rRNA3 -SDmRNA


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3


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3.1


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3.1.1


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  • 3

DNA


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1DNA

oriDNA

pMB1p15AColE1:1020

pSC101:5


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E1R


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2

promotorRNADNA


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RNA

69bp

mRNAmRNA


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RNAmRNA


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terminatorRNADNA

RNAmRNAmRNA


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3

SDSDmRNAmRNA55

SD


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DNAShine-DalgarnoSDSD16S rRNAmRNA

mRNAmRNA SD16S rRNASD16S rRNA

mRNASDSD

SD5 AGGAGG 3 ,GGAG


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SDSD68bp7bp

SDSDAAAAUUUUCCCCGGGG5025

SDSDSD


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AUGATGMETGUGUUG

mRNA

RF1RF2 RF1UAAUAG RF2UAAUGAUAA


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UAAU

mRNA


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3.1.2

LacTaclac

PLPRPLPR

T7T7


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3.1.3


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RNA

DNARNA


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*


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SD

SD


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DNADNA


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N1530signal peptidesN


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3.1.4

  • tRNA

  • mRNA


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3.2.1

yeast

  • 1996


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3.5.1

DNAARSCENTEL

  • DNA


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12kbTATATATAPGKGAPDH

N1730

mRNA3mRNA3 mRNA


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3.2.2

  • yeast replicating plasmidYRP

DNA200

  • yeast integration plasmidYIP

DNADNADNA


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  • yeast centromeric plasmidYCP

12

  • yeast artificial chromosomeYAC

DNA

SUP4SUP4YAC200800kb


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3.2.3

*


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AOX1 AOX1AOX1AOX1his


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-

-1-


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3.2.4


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3.3 *

mRNA

6.3.6.1


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poly(A)


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SV40

100200bpSV40


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  • poly(A)

RNApoly(A)DNApoly(A)1130AAUAAApoly(A)GUU

poly(A)SV40237bpBamH I-Bcl Ipoly(A)


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poly(A)mRNA mRNA5 3 SV40cDNA


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3.3

  • SV40

  • BPV

  • EBV

  • adenovirus

  • retrovirus


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3.4

CHO-K1COS


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  • CHO-K1

CHO-K1dhfr10g/ml

CHO-K1

tPACHO


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  • COS

CV-1SV40T

COS

SV40

COSCOS


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Sp2/0NSOJ558LIgtPA


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3.6 *

1

2


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3

4

N-O-


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3.7

3.7.1


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3.7.2

TiT-DNA

Ti

Ri


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  • Ti

Ti200250kb90103150103KDDNA Ti

IAA

Ti


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TiVir region T-DNAVirT-DNA

octopinenopalineagropine3


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  • CaMV35Snosocs


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mRNAmRNA

TGATAA

  • T-DNA

T-DNADNAtransfer-DNATi1025kbT-DNAT-DNA25bpT-DNAT-DNA1~2


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TiVirT-DNAT-DNAT-DNA25bpT-DNAT-DNADNATi


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mRNAmRNA

  • *

  • selectable marker genes


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  • neomycin phosphotransferase , NPT neo

  • dihydrofolate reductase, DHFRdhfr

  • hygromycin phosphorransferase , HPThpt

  • phosphinothricin acetyltransferasebar

marker genes


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  • reporter genes

  • --glucuronidase, GUS

  • luciferase, LUC

  • chloramphenicol acetyltransferase, CAT


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3.7.3


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3.7.4

  • 53


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4

4.1

mRNA

mRNA

Northern


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4.1.1

SouthernNorthernmRNA

Northern Southern

  • RNA

  • mRNA

  • RNA

  • Northern

RT-PCR


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4.1.2

-guscatnpt-


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  • cat

50S

catCoAcatcat

catCoACoASHGDTNB


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  • gus -

gus--gusgusgusgus


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6-Mg2+ATPO2

ATP+ O2 CO2+AMP+PPi+

RCHO+ O2+FMNH2 RCOOH+H2O+FMN+


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  • dhfr

dhfr


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4.1.3

Western

100pg


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  • enzyme-linked immuno sorbent assay, ELISA

ELISA1pgELISAELISA

ELISA


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  • Western

Western50ng15ng

SDS-


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Western

SDS-


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  • RIA

RIA1pg

RIA

RIA

RIA

RIA

RIA


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4.1.4


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4.2

6.4.2.1


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4.2.2

g

100,000g

RCF=11.18(N/1000)2r

RCFg

Nr/min

rcm


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4.2.3


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4.2.4


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4.2.5

polyacrylamide gel electrophoresis, PAGE

1kb DNADNA

acrylamideN,N-APN,N,N, N-TEMEDN,N-


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  • 0.1DNA1000bp1bp

  • 10gDNA1cm1cm

  • DNA

Native-PAGE

PAGE

SDS-PAGE


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