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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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mRNA


  • RNAmRNA mRNAmRNA

  • hnRNA53mRNAmRNA


1

1.1

lactrpPR PL tac

T7


1.2 mRNA

mRNA

mRNA

mRNA


1.3 mRNA

mRNA

  • AUG(ATG)

  • SD16S rRNAAGGAGG4

  • SD39

  • mRNA


major codon

rare codon

mRNA


1.4


1.5

DNA

RNA


2

2.1

promoterRNADNARNA1035TATA

*


2.1.1

  • CATA/G

  • Pribnow 10bpTATA10

  • Sextama35bpTTGACA35


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.


2.1.1

rRNA

mRNA

tRNA


rRNArRNA

UCE

180190bp


TATAHognessATDNA2030bp

CAAT75bpRNA

GC100300bp


tRNA5SRNA

U6RNA7SKRNA


2.1.3

RNA

RNA

(2)( 2 )


sfactor:


2.1.4

PCR


2.2

enhancerDNA


2.3

RNA


()RNARNA

U


5090bp GCRNAURNA

2.0105NTPNTP RNA


RNARNAATP53RNARNA3OHRNARNA


2.4

attenuatormRNA

trp

trp412341-23-42-3

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


Low Trp

High Trp


2.5

  • insulator


2.6

RNAantisence RNAmRNARNARNADNAmRNARNAantisence techology

RNADNA

DNA


  • RNARNARNADNADNA

  • RNADNA

  • RNAmRNA5

  • mRNApoly(A)mRNA

  • mRNA


  • mRNA5 SDmRNA

  • mRNA5

SDSD sequenceShine-DalgarnomRNAAUGAGGAGGG16S rRNA3 -SDmRNA


3



3.1


3.1.1


  • 3

DNA


1DNA

oriDNA

pMB1p15AColE1:1020

pSC101:5


E1R



2

promotorRNADNA


RNA

69bp

mRNAmRNA


RNAmRNA


terminatorRNADNA

RNAmRNAmRNA


3

SDSDmRNAmRNA55

SD


DNAShine-DalgarnoSDSD16S rRNAmRNA

mRNAmRNA SD16S rRNASD16S rRNA

mRNASDSD

SD5 AGGAGG 3 ,GGAG


SDSD68bp7bp

SDSDAAAAUUUUCCCCGGGG5025

SDSDSD


AUGATGMETGUGUUG

mRNA

RF1RF2 RF1UAAUAG RF2UAAUGAUAA


UAAU

mRNA


3.1.2

LacTaclac

PLPRPLPR

T7T7


3.1.3


RNA

DNARNA


*


SD

SD


DNADNA


N1530signal peptidesN



3.1.4

  • tRNA

  • mRNA


3.2.1

yeast

  • 1996


3.5.1

DNAARSCENTEL

  • DNA



12kbTATATATAPGKGAPDH

N1730

mRNA3mRNA3 mRNA


3.2.2

  • yeast replicating plasmidYRP

DNA200

  • yeast integration plasmidYIP

DNADNADNA


  • yeast centromeric plasmidYCP

12

  • yeast artificial chromosomeYAC

DNA

SUP4SUP4YAC200800kb


3.2.3

*


AOX1 AOX1AOX1AOX1his


-

-1-


3.2.4


3.3 *

mRNA

6.3.6.1


poly(A)


SV40

100200bpSV40


  • poly(A)

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

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


poly(A)mRNA mRNA5 3 SV40cDNA


3.3

  • SV40

  • BPV

  • EBV

  • adenovirus

  • retrovirus


3.4

CHO-K1COS


  • CHO-K1

CHO-K1dhfr10g/ml

CHO-K1

tPACHO


  • COS

CV-1SV40T

COS

SV40

COSCOS


Sp2/0NSOJ558LIgtPA


3.6 *

1

2


3

4

N-O-


3.7

3.7.1


3.7.2

TiT-DNA

Ti

Ri


  • Ti

Ti200250kb90103150103KDDNA Ti

IAA

Ti


TiVir region T-DNAVirT-DNA

octopinenopalineagropine3


  • CaMV35Snosocs


mRNAmRNA

TGATAA

  • T-DNA

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


TiVirT-DNAT-DNAT-DNA25bpT-DNAT-DNADNATi


mRNAmRNA

  • *

  • selectable marker genes


  • neomycin phosphotransferase , NPT neo

  • dihydrofolate reductase, DHFRdhfr

  • hygromycin phosphorransferase , HPThpt

  • phosphinothricin acetyltransferasebar

marker genes



  • reporter genes

  • --glucuronidase, GUS

  • luciferase, LUC

  • chloramphenicol acetyltransferase, CAT


3.7.3



3.7.4

  • 53


4

4.1

mRNA

mRNA

Northern


4.1.1

SouthernNorthernmRNA

Northern Southern

  • RNA

  • mRNA

  • RNA

  • Northern

RT-PCR


4.1.2

-guscatnpt-


  • cat

50S

catCoAcatcat

catCoACoASHGDTNB


  • gus -

gus--gusgusgusgus


6-Mg2+ATPO2

ATP+ O2 CO2+AMP+PPi+

RCHO+ O2+FMNH2 RCOOH+H2O+FMN+


  • dhfr

dhfr


4.1.3

Western

100pg



  • enzyme-linked immuno sorbent assay, ELISA

ELISA1pgELISAELISA

ELISA


  • Western

Western50ng15ng

SDS-


Western

SDS-


  • RIA

RIA1pg

RIA

RIA

RIA

RIA

RIA


4.1.4


4.2

6.4.2.1


4.2.2

g

100,000g

RCF=11.18(N/1000)2r

RCFg

Nr/min

rcm


4.2.3


4.2.4


4.2.5

polyacrylamide gel electrophoresis, PAGE

1kb DNADNA

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


  • 0.1DNA1000bp1bp

  • 10gDNA1cm1cm

  • DNA

Native-PAGE

PAGE

SDS-PAGE


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