华东理工大学：基因工程：jygc 6

3t/ ?<?? [? 3t/ ?<?? [? yy? ?ê ?yvD fòZ ?ê ?yvD yy? 5 2 3 4 1 6 7 8 9 ×F DNA/ ?Dyy?￥'àQ / ?Dyy?￥'àQ ×F DNA/ ?Dyy?￥'e ? / ?Dyy?￥'e ? ×F DNA/ ? ?3￥'Hq / ? ?3￥'Hq ×F DNA/ ?￥?TV? / ?￥?TV? "￥y￥ X HDyó o￥y "￥y￥ X HDyó o￥y ?÷yv?? )?￥Vr ?÷yv?? )?￥Vr ?÷y? )?￥Vr ?÷y? )?￥Vr ?÷y??t%?￥Vr ?÷y??t%?￥Vr ?÷yVrát￥s ?B? ?÷yVrát￥s ?B? D yy? )￥L.?×??#| yy? )￥L.?×??#| 6 ?÷yv?? )?￥Vr ?÷yv?? )?￥Vr C v?? )yy? )￥y| { v?? )yy? )￥y| { B ?÷yv?? )?úrVr￥e ? ?÷yv?? )?úrVr￥e ? A v?? )T1Vr?÷y s8 )￥+? v?? )T1Vr?÷y s8 )￥+? E ?¨×Fv?? ) 3á|O? í ?¨×Fv?? ) 3á|O? í A v?? )T1Vr?÷y s8 )￥+? v?? )T1Vr?÷y s8 )￥+? 6 ?÷yv?? )?￥Vr ?÷yv?? )?￥Vr v?? )Vr?÷y￥a ] v?? )Vr?÷y￥a ] ?yF??
μ 4405? 7b? O ?yF??
μ ? 7b? O y X HVr"d? ??? y X HVr"d? ??? N2á ?a?!e?a?TZLaL.×? N2á ?a?!e?a?TZLaL.×? $ ?S FDA?1??￥yy? s8 3t $ ?S ?1??￥yy? s8 3t A v?? )T1Vr?÷y s8 )￥+? v?? )T1Vr?÷y s8 )￥+? 6 ?÷yv?? )?￥Vr ?÷yv?? )?￥Vr v?? )Vr?÷y￥  ] v?? )Vr?÷y￥  ] ?C?? 3t?aé￥ˉ??? ?C?? 3t?aé￥ˉ??? ?C?? 3t?aé￥? eFy"d ?C?? 3t?aé￥? eFy"d =÷??a ??3 bW`???￥s÷?a =÷??a ??3 bW`???￥s÷?a %?é=cμ? ?N￥=y í %?é=cμ? ?N￥=y í B ?÷yv?? )?úrVr￥e ? ?÷yv?? )?úrVr￥e ? 6 ?÷yv?? )?￥Vr ?÷yv?? )?￥Vr ?0 ??0 ?82?ê? ?82?ê? á ?0 á ?0 é ì J ? é ì J ? ?0 ?0KD  ?￥? ?0KD  ?￥? "￥y "￥y E E A E E ?0 ?0 A ?M 7 ?M 7 Bal31 ?3 ?3 "￥y "￥y ?0 ?0￥?ê Ap r r ori galKpKO1 ?? á ?0 ?? á ?0?¨T6E^ {
|? avl￥ DNA? ?¨T6E^ {
|? avl￥ ? X H??0?é ì pKO1  X H??0?é ì  s8%??8 DNA ￥ galEagalTDé s8%??8 Dé ì áy galk￥Vrát ó?T¨
 ì áy ￥Vrát ó?T¨
 V|?!?￥???3?￡? íté V|?!?￥???3?￡? íté ? galE + agalT + agalK - ￥v?? ) s8 )é ? - ￥v?? ) s8 )é cμ?÷?0?￥×F X H cμ?÷?0?￥×F X H ?0 ?0￥y -35 u?  u?  -10 u?  u?  P λL P recA P trp P lac P traA P tac ?0 T T G A C A G A T A C T T T G A T A T A T A A T T T G A C A T T A A C T T A G A C A T A A T G T T T T A C A T A T A A T T T G A C A T A T A A T P tac = 3 P trp = 11 P lac ?0 ?0￥ V e? P ??0 P lac ￥ V e?
 ￥ V e?
 O P O úr c úr c E3?a E3?a o? o? ? sd -β-D- ; }??
IPTG
 ? sd ; }??
7 3?￥ P lac D e?u O lac 7 3?￥ la D e?u } óB
 àμo?t } óB
 àμo?t i H
???:0)? i H
???:0)? ? ￡ü c ? ￡ü c ? ￡üVr
o?t V[ P ? ￡üVr
o?t V[ P ?0 P lac o?￥ cv? ?0 o?￥ cv? 4ú 4ú ?0 ?0￥ V e? P lac la ??0 P lac ￥ V e?
 ￥ V e?
 O P lac la O úr c úr c ?$}? ?$}? 7 3?￥ P lac 3?í?μ 7 3?￥ la 3?í?μ }?y0
CAP
2? }?y0

2? u
 cAMP CAP
CAP  ? ￡ü c ? ￡ü c 2??0 e?u
é72??0 e?u
é7é P lac o?￥ cb?$ é o?￥ cb?$ }? P cAMPh 
9?E }? P h 
9?E 3P lac o?￥ cbyN
 o?￥ cbyN
 yy?? P¨￥?? yy?? P¨￥?? 0 (1 F?$}?E3￥ 0 (1 F?$}?E3￥ lM?
' P lac UV5 lM?
' lac CAP cAMP cAMPi?? i?? P lac UV5 lac O úr c úr c ?0 ?0￥ V e? P trp trp ?? ??0 P trp ￥ V e?
 ?? ??0 ￥ V e?
 O trp tr "??? ? "? ?? ? ?? ??0 P trp s?? ? -E3 ?? ??0 trp s?? ? ?aˉ?t￥E3
 c?? ?aˉ?t￥E3
 c??  ?b?!"d??"?? ?  ?b?!"d??"?? ? ? ￡ü c ? ￡ü c ?F? 3- )d ?
IAA

 ?F?  )d ?
L Vμr13"E3]?T¨b L Vμr13"E3]?T¨b ?è￥% )?!8"?
"? ?è￥% )?!8"?
"? ?? ? ^ ?4￥
yNyy? ?? ? ^ ?4￥
yNyy? ?aa@F IAAo? P trp o?￥" ?aa@F o? trp o?￥" y￥Vr y￥Vr ?? ? ?? ? F 3- )d ? F  )d ? úr c úr c E3?a E3?a
IAA

O trp tr P trp trp úr c úr c O trp tr P trp trp ?0 ?0￥ V e? λ ` )8?0 P λL ￥ V e?
 ` )8?0 ￥ V e?
 ` )8?0 P L s CIE3?aE ` )8?0 s E3?aE 3
?4°¤o? e?by 3
?4°¤o? e?by y??è P¨? ù??￥ cIl y??è P¨? ù??￥ l My cI 857 e? P L b cI 857 E3? My e? E3?  42￥ H >? ?
 P L L Vo?  ￥ H >? ?
 L Vo? "￥y￥Vrb?v?% ) "￥y￥Vrb?v?% ) ?!7?á ? 6?dè ?4
y ?!7?á ? 6?dè ?4
y Nè P¨B? ?é ì e?"d
 Nè P¨B? ?é ì e?"d
 ¨?? ?W¤ e?"￥yVr ¨?? ?W¤ e?"￥yVr P trp trp A B cI 857 P L "￥y "￥y E3T¨ E3T¨ P trp trp A B P L Vr Vr ?? ? ?? ? ??0 <? c??￥A1? <? c??￥A1? ?÷y<?0￥ e?/Vr
?^? 3 cVhC`
' RNA ? ?á V??02?? cé ì  #í￥ DNA? 
??é?B￥ mRNA
?t Vé ct￥á 3?v? ??Y? ÷y￥Vr
ey?/
  cáté
 RNA ? ? cBs0 mRNA ?3￥ HWüM?9F
?÷y ' &￥ cr q/?
?T?÷y/3?¤μ8 ￥ ?×1y DNA??u×
?ê4?S :y?ˉ?02?
5 RNA ? ?N)￥ c V???é ì￥ˉ?# ? 3 t??
 .à?á×Fé ì￥?×??
Vé￥ mRNAaa?á 3v í¨￥?aé
9Fy? )íì￥? h{
÷1?×￥ ^
Vé￥ ctaa???? ?X￥=)2
V7vv???÷ yI ?át￥Jrr q ?÷y<?0￥ e?/Vr
?^? 3 cVhC` ' ? ?á V??02?? cé ì  #í￥ ? 
??é?B￥ 
?t Vé ct￥á 3?v? ??Y? ÷y￥Vr
ey?/
  cáté
 ? ? cBs0 ?3￥ HWüM?9F
?÷y ' &￥ cr q/?
?T?÷y/3?¤μ8 ￥ ?×1y ??u×
?ê4?S :y?ˉ?02?
5 ? ?N)￥ c V???é ì￥ˉ?# ? 3 t??
 .à?á×Fé ì￥?×??
Vé￥ aa?á 3v í¨￥?aé
9Fy? )íì￥? h{
÷1? Vé￥ ctaa???? ?X￥=)2
V7vv???÷ yI ?át￥Jrr q ??0 <??0￥ê4D P¨ <??0￥ê4D P¨ "-?÷yVré ì?è¨￥? ?0 ^ ?1v?? ) rRNA?:0 ￥ rrnT 1 T 2 [# T7 ` )8 DNA ￥ Tφb ?Bt??T¨?D￥??0
Yè V[?¨= 8??01 ó￥+ y2 
[9< c??T¨ ??09 V[`?0*"
YV + y￥?é ìV% ) ` )8y FDNA? X H?ê "-?÷yVré ì?è¨￥? ?0 ^ ?1v?? ) ?:0 ￥ [# ` )8 ?Bt??T¨?D￥??0
Yè V[?¨= 8??01 ó￥+ y2 
[9< c??T¨ ??09 V[`?0*"
YV + y￥?é ìV% ) ` )8y ? X H?ê pCP1 Ap r r ori Tc r r ?ê Ap r aTc s ￥?0 ?ê ?82?ê? ?82?ê? ?÷yv?? )%?￥ úrVr??| %? c?￥ ? q
7O?v? ?D mRNA￥Jr Sr q áMM1bv ?? )%?2?]￥ mRNAs0 μ?]￥Jrr q
 ? ì- W￥μYμ H Vúr ??b mRNAJr￥ Sr q?1? 5‘  ￥2?  ? %?
?1?82?ê?
RBS
 ?÷yv?? )%?￥ úrVr??| %? c?￥ ? q
7O?v? ?D ￥Jr Sr q áMM1bv ?? )%?2?]￥ s0 μ?]￥Jrr q
 ? ì- W￥μYμ H Vúr ??b Jr￥ Sr q?1? ￥2?  ? %?
?1 ?82?ê?
?82?ê? ?82?ê? v?? )?82?ê? ?/  1?+?21 í
 ê?Jr S á ?0 3￥ 6-8?? ??  5’ UAAGGAGG 3’
' Shine-Dalgarno
SD
? 
 ?YV MYv?? )?8le? ￥16S rRNA 3’ u× 3’ AUUCCUCC 5’iD-B?2?
| mRNA?ê??8 
V7?Jr
Jr S á ?0
v?? ) 'v?sy[ AUGT1 O￥ Sê?
?μty9 P¨ GUG UUGT1Jr S á ?0
SD? DJr S á ?0-W￥  ?#aF?
yI ?u 5’  ?? á ?0￥a?  v?? )?82?ê? ?/  1?+?21 í
 ê?Jr S á ?0 3￥ ?? ??  ’
? 
 ?YV MYv?? )?8le? ’ iD-B?2?
| ?ê??8 
V7?Jr
Jr S á ?0
v?? ) 'v?sy[ Sê?
?μty9 P¨  T1Jr S á ?0
? DJr S á ?0-W￥  ?#aF?
yI ?u  ?? á ?0￥a?  ?82?ê?￥2 ?82?ê?￥2 ?82?ê? ?82?ê? ?82?ê??÷yVr￥?Y ?82?ê??÷yVr￥?Y SD? ￥?Y
 ? ￥?Y
 B? ? a
 mRNAD?8￥2??<
Jr￥ Sr qüú
7??2???1| %? SD? D 16S rRNA￥a o??
?[ GGAG 1?a? -1×1b ?y7 y
  ? 1?a???B?D? C T
 (??áJrr qv? ?? B? ? a
 D?8￥2??<
Jr￥ Sr qüú
7??2???1| %? ? D o??
?[ 1?a? -1×1b ?y7 y
  ? 1?a???B?D? 
 (??áJrr qv? ?? ?82?ê? ?82?ê? ?82?ê??÷yVr￥?Y ?82?ê??÷yVr￥?Y SD? D S á ?0-W￥? ￥?Y
 ? D S á ?0-W￥? ￥?Y
 SD? /3￥a?1 AAAA UUUU
Jrr qKú
7 CCCC GGGG￥Jrr q5sY ^Kú′￥ 50%? 25%b? # AUG￥-??a?zJr S9μ?Y
?v?? ) β-? ? ?￥ mRNA7y
??ê? KD￥aF? ^ UAU CUU
?T¨ UUCaUCA AGG|}-
5 ?￥Vr ￡ü? 20  ? /3￥a?1 
Jrr qKú
7  ￥Jrr q5sY ^Kú′￥ ? b? # ￥-??a?zJr S9μ?Y
?v?? ) ? ? ?￥ 7y
??ê? KD￥aF? ^ 
?T¨  |}-
5 ?￥Vr ￡ü?  ?82?ê? ?82?ê? ?82?ê??÷yVr￥?Y ?82?ê??÷yVr￥?Y SD? D S á ?0-W￥  ?￥?Y
 ? D S á ?0-W￥  ?￥?Y
 SD? D S á ?0-W￥ú?  ?￡  mRNA?8 ?êa
Jr S á ?0 AUG?z)??8ˉ?t2?￥ P ê
? ^Jr?￥-4Hqb?f ?/
 SD? ê? AUG --v?t?a)
NW?? B?aB?a
 ( ??áJr Sr q?]?￥?? ? D S á ?0-W￥ú?  ?￡  ?8 ?êa
Jr S á ?0 ?z)??8ˉ?t2?￥ ? ê? --v? t?a )
NW?? B?aB?a
 ( ??áJr Sr q?]?￥?? ?82?ê? ?82?ê? ?82?ê??÷yVr￥?Y ?82?ê??÷yVr￥?Y  S á ?0#a??? á ?0￥?Y
  S á ?0#a??? á ?0￥?Y
 v?? )?￥ S tRNAs0 V[] H MY AUGaGUG? UUG ?? S á ?0
? MY? qi?M]
Yè GUG1 AUG￥50% 7 UUGo# AUG￥25%b"N-?
V AUG 7 S￥-+? á ?0a ? 9à1×1
à ?B? ??D mRNA￥5’  dI ?u? ??ì2
?5L??×??N3/"?8 ￥??ê "-<W¨??÷yVr￥ v?? )Vr?é ì 
 (cμ D?0 ?÷M]￥?82?ê ?? 
? ?W? ^KD￥ v?? )?￥ S s0 V[] H MY ? ?? S á ?0
? MY? qi?M]
Yè o# 7 S￥-+? á ?0a ? 9à1×1
à ?B? ??D  dI ?u? ??ì2
?5L??×??N3/"?8 ￥??ê "-<W¨??÷yVr￥ v?? )Vr?é ì 
 (cμ D?0 ?÷M]￥?82?ê ?? 
? ?W? ^KD￥ á ?0 á ?0 3t8 á ?0￥ê1? 3t8 á ?0￥ê1? ?]￥ 3t
 .à]? 3t? ]￥?aéI ?y
ei á ? ?]￥ 3t
 .à]? 3t? ]￥?aéI ?y
ei á ? 0 P¨? qi?M]
 μB?￥ê1?
 %?y í ^
 0 P¨? qi?M]
 μB?￥ê1?
 %?y í ^
 3tyF?￥ac  ?c AT￥ 3t
?? y DNA ` )8 ?c ￥ 3t
?? y ` )8 φ9
yF?
 á ?0??ê ￥ U? AC￥? q?ú
7 GC 9
yF?
 á ?0??ê ￥ ? C￥? q?ú
7 ~?￥ 3t
? y ? )
yF?
??ê cμ G C￥ei á ?0 ~?￥ 3t
? y ? )
yF?
??ê cμ  ￥ei á ?0 ] 90%[ ￥ 'a ] [ ￥ 'a ] á ?0DQ á ?0MoT¨￥1?? ???<? p á ?0DQ á ?0MoT¨￥1?? ???<? p %= tRNA￥c  %= ￥c  ? GGGaCCCaGCGaGGCaAAAaUUUaAUAaUAU? P¨ 
? P¨ 
? GUGaCACaUCGaAGCaACAaUGUaAUCaUUG? P¨
? P¨
á ?0 á ?0 á ?0ê1??÷yVr￥?Y á ?0ê1??÷yVr￥?Y ??e? 3t??? 3ty F? á ?0￥ P¨? q μ?v? ??e? 3t??? 3ty F? á ?0￥ P¨? q μ?v? v￥μs?
yN?÷y- ^ ú???tyv?? )?ú v￥μs?
yN?÷y- ^ ú???tyv?? )?ú rJr￥B?×1y í ^ á ?0￥ ??ê4bB?7y
μ ?| { rJr￥B?×1y í ^ á ?0￥ ??ê4bB?7y
μ ?| { V[ P?÷y ￥ á ?0v?? )%?¤KDVr
 V[ P?÷y ￥ á ?0v?? )%?¤KDVr
 ?÷y??? ?÷y??? ]?VrM1 tRNAI ?y ]?VrM1 I ?y á ?0 á ?0 á ?0ê1??÷yVr￥?Y á ?0ê1??÷yVr￥?Y ?vv?? ) á ?0￥ê1?? p
 !9÷D?÷y?? a R￥M?ei á ?0
×F|O? ía?? í[# 3é í v?? )?￥úrVr (?¨ ??ZE ?vv?? ) á ?0￥ê1?? p
 !9÷D?÷y?? a R￥M?ei á ?0
×F|O? ía?? í[# 3é í v?? )?￥úrVr (?¨ ??ZE ?÷y??? ?÷y??? á ?0 á ?0 á ?0ê1??÷yVr￥?Y á ?0ê1??÷yVr￥?Y ?*tcμ??~ á ?0? ??BaC? q?úa7' &s 0 ??v￥?÷y7y
5ê4M1 tRNAI ?y]? X HVr ￥| {?1μ ?b è?
|U ?e cDNA￥412? á ?0?
c μ 22?ú? ? á ?0
? 7? AGGa2? AGA
7v?? ) s8% ? tRNA AGG ? tRNA AGA ￥~??b1 4ú|U ?e cDNAv? ? )?￥úrVr
|v?? )￥? ? tRNAI ?y X H 6B? úVr￥é ì b?Ny￥v? ? ) ?é ì"dμr13"  s8 %?÷yúrVr￥??T¨ ?*tcμ??~ á ?0? ??BaC? q?úa7' &s 0 ??v￥?÷y7y
5ê4M1 I ?y]? X HVr ￥| {?1μ ?b è?
 |U ?e ? á ?0?
c μ ? ú? ? á ?0
?
7v?? ) s8% ? ￥~??b1 4ú|U ?e v? ? )?￥úrVr
|v?? )￥? ? I ?y X H 6B? úVr￥é ì b?Ny￥v? ? ) ?é ì"dμr13"  s8 %?÷yúrVr￥??T¨ ]?VrM1 tRNAI ?y ]?VrM1 I ?y é ì J ? é ì J ? é ì J ?% ) 3é}?￥?Y é ì J ?% ) 3é}?￥?Y "- L i ú<W P¨￥Vr ?é ì ??v?? )%? Vr ?? .à $? J
é ì￥ ?9 V?Yè? 3 s8%￥ ? 3 éù=
7N H? ^% ) 3 ?}? K? 9￥¨ bé ìs0￥V9 2[#a"￥y￥úrVr ] A??Y s8%￥ 3é}?
é 7?á×Fé ì￥?×??[#"￥y?4Vr ￡ü￥/? 3 %  ?45￥B?μr| { ^|×Fé ì￥ ?9,? V e?￥ E? "- L i ú<W P¨￥Vr ?é ì ??v?? )%? Vr ?? .à $? J
é ì￥ ?9 V?Yè? 3 s8%￥ ? 3 éù=
7N H? ^% ) 3 ?}? 2[#a"￥y￥úrVr ] A??Y s8%￥ 3é}?
é 7?á×Fé ì￥?×??[#"￥y?4Vr ￡ü￥/? 3 %  ?45￥B?μr| { ^|×Fé ì￥ ?9,? V e?￥ E? é ì J ? é ì J ? é ì ?9 H?￥ e? é ì ?9 H?￥ e? pCP3?μB?? Vo??￥ˉ?0 ?μB?? Vo??￥ˉ?0 pCP3 P L MCS oriAp r r  28￥ H
 ??%￥é ì J ?1 60 ￥ H
 ??%￥é ì J ?1  42￥ H
 J ?á ?9à 300 - 600 ￥ H
 J ?á ?9à N?/
 s8%??8 ￥ CI N?/
 s8%??8 ￥ yVr￥? ù??E3?a > yVr￥? ù??E3?a > yN
¨B? m ] H e?é ì J yN
¨B? m ] H e?é ì J  ??y￥Vr  ??y￥Vr C v?? )yy? )￥y| { v?? )yy? )￥y| { 6 ?÷yv?? )?￥Vr ?÷yv?? )?￥Vr d8?s÷?a￥Vr d8?s÷?a￥Vr s ??s÷?a￥Vr s ??s÷?a￥Vr μ??s÷?a￥Vr μ??s÷?a￥Vr * ?s÷?a￥Vr * ?s÷?a￥Vr ???s÷?a￥Vr ???s÷?a￥Vr ?a ? F??J?Vr"d￥y ?a ? F??J?Vr"d￥y d8?s÷?a￥Vr d8?s÷?a￥Vr d8#?é d8#?é t 3éHq/
v?? ) ? ??+ y￥ 3tvs0
 ? ìá á1" %=
$ U??í ? ]2
?? ￡? ·?￥2?1d8
Inclusion Bodies
IB
b?c?aé￥d 8n? 3écμ? ? ? ?t ?!￥v?? )%?
??t ? ? ? ?t ???￥?aéaa ?T > ??+?? 3t??
V 7" ??d8b?úrVré ìy￥v?? )y? )v ?? d???￥]÷s÷?aé
a ?B?f ?/9[d8￥? T i?% )%=b"N-?
d8??cμ  ￥ DNAaRNA? ??d?as0 t 3éHq/
v?? ) ? ??+ y￥ 3tvs0
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 S S S S C N O?a ? O?a ? C peptide A peptide B peptide M R R K R CNBr R S S S S C N N C S S S S R ?t ? B ?t ? B y?? 22ê ￥ Arg?? 29 y?? ê ￥ ?? ê ￥ Lys?? z|?￥e ê ￥ ?? z|?￥e y
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