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Expression of human serum albumin in Kluyveromyces lactis

人血清白蛋白在乳酸克鲁维酵母中的表达



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ChineseJournalofBioprocessEngineering
Vol.10No.5
Sep.2012
doi:10.3969/j.issn.1672-3678.2012.05.011
NOPH
:2011-03-01
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à—‚ƒ–Ò„
(Kluyveromyceslactis,K.lactis)GG799
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èÿ“^z{ËV
(SDS PAGE)
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Westernblot
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Å%ìh҄Ƃ

ã
(YNB)
Ï
HSA
Ã
K.lactisGG799
vØÿÓÔÕ

Ö×ØÙ
,HSA
‡™Ã
K.lactisGG799
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:1672-3678(2012)05-0055-06
ExpressionofhumanserumalbumininKluyveromyceslactis
FULili1,GUANBo1,LEIJianyong2,DUANZuoying1,CHENYun2,JINJian2,LIHuazhong1
(1.KeylaboratoryofIndustrialBiotechnologyoftheMinistryofEducation,SchoolofBiotechnology,JiangnanUniversity,
Wuxi214122,China;2.SchoolofMedicine,JiangnanUniversity,Wuxi214122,China)
Abstract:Thesecretoryexpressionofhumanserumalbumin(HSA)inKluyveromyceslactisGG799was
investigated.TheHSAgenewasamplifiedbyPCRusingpPIC9kHSAastemplateandprimerswithXho
ⅠandNotⅠrestrictionsites,respectively,andthenclonedintotheexpressionvectorpKLAC1byXhoⅠ
andNot.RecombinantplasmidpKLAC1HSAwaslinearizedbySalⅡandtransformedintoKluyveromyces
lactisstrainGG799byelectroporation.RecombinantswerescreenedbyYCBplatescontaining5mmol/L
acetamide.ThegenomicDNAoftransformantswasisolatedtoidentifythepositiveonesbyPCR.The
transformantswerefermentedinshakeflaskandtheexpressionproductswereconfirmedbySDSPAGE
andWesternblot.TheefectofYNBinculturemediumonexpressionofHSAinKluyveromyceslactis
werealsoinvestigated.TheresultsindicatedthatHSAwasexpressedbytheKluyveromyceslactisGG799
expressionsystemandtheaverageyieldwas81μg/mL.
Keywords:humanserumalbumin;Kluyveromyceslactis;secretedexpression
  
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6m
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,PCR
[
:95℃
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wÚô
;72℃

5min。
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1 
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Table1 Sequencesofprimersintheexperiments
yv
5′—3′
KH1
GCCTCGAGAAAAGAGATGCACACAAGAGT
GAGGTTGCTC(
tj$è
XhoⅠF€‚A,¨ ,
蔓F
Kex
F€‚A
KH2 GTGGCGGCCGCTTATAAGCCTAAGGCAGCTTG(
tj$è
NotⅠF€‚A)
KJD1 TACCGACGTATATCAAGCCCA
KJD2 ATCATCCTTGTCAGCGAAAGC
 
!
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è
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»›-.
,16℃
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HSA
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Fig.1 SchematicofexpressionvectorpKLAC1HSA
´7¤

  
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。SalⅡF€ pKLAC1HSAr
pKLAC1(
<“-Û

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PCR
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mmol/L
¥¦ˆ2
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30℃
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H
3d。
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,30℃
GH
24h,
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Ižˆ

J,­2Ižˆè£§
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r
KJD2
èåL

qnG9*
K.lactisGG799pKLAC1HSA
Jr
K.lactisGG799pKLAC1。
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
K.lactisGG799
c…2)ÃÖº
r
Westernblot
²³
  
¾­
YCB
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5mLYPD
G
HI
,200r/min、30℃
GH
24h,
²Ý­
100μL
YPD
z*©±z)
10mLYPG
r
10mLYPG+
134%YNB(
"M
Invitrogen
mÌ2(1˜…Ÿ
÷
),
JG±Iè
0h,
ÈÉ
6h
­Ù

¾å½LǺ
s
A600,‡­¯’dgÁ{I¶X0 Uk¦
ˆß›)Ã
(SDS PAGE)
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ûÇ
,100V、100min
Gì)Â
XCDEÒ
,TBST(pH75Tris HCl10mmol/L,
NaCl150mmol/L,Tween 20005%)
_´4“Ç
rl
5%
¨e5‚c—Ú67
2h,
ºÇrl67©
½L
(1∶2000)
2ÿ
HSA
ÿ,
(HRP
Q=

c—Ú
3o
2h,TBST
“cÇd
DAB
ñÓ
[7]。
124 
P6Y»`Öº
  
²þ¤2Y¶S
K.lactisGG799pKLAC1HSA
ÀGHI
YPG+134%YNB
cè7GH
30
7

­
GH
48h
201¯’©Jgà´µ2
HSA
è}"

Westernblot
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J+èPQMNY¶S…
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2P6Y»`

2 
xy%z)
21 HSA
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pKLAC1HSA
^•Ú
  
J
pPIC9kHSA
裧

Jp;
XhoⅠP
NotⅠF€‚A2åL6m HSAIž,yUÞ



1—DNAMarkerDS2000;2—PCR
>L
f
2 HSA
J¬^µ¶
Fig.2 AmplificationofHSAgene
  
²Ý-6m2
HSA
Iž0=P
pKLAC1

XhoⅠr NotⅠ&F€,-.0=d4±、G9。
G9*hž³GHÇ,­p¡d
PCR
PF€û
»

yUÞ



Þ

…ð‡ˆp¡
pKLAC1
HSA
þ¤ÍÄ

-v`p¡dº¿

yñß
HSA
Iž¿3ÆÍÄ

f
3 
«´¼·
pKLAC1HSA
^9Ù
Fig.3 Identificationofrecombined
plasmidpKLAC1HSA
75 
!

# ì""y

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22 K.lactisGG799
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78
  
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pKLAC1HSA

SalⅡGF€$`9,>TN80r28kb20=

<“-Û
pKLAC1
$`9F>TN
62
r
28kb
20=
),

4(a)、
Þ
4(b)


)2G9
K.
lactisGG799
Ž]ü~

¾­
YCB(
—
5mmol/L
¥¦ˆ

c§¯2GS¿ž³GHÇ

,­1˜I
žˆè£§d
PCR
û»

žÁ20=ðl
LAC4
zº*ö

AˤåL
KJD1、KJD2
6mµž
19
kb
0=

F…ßÁ2IžÍÄâ©)
K.lactisGG799
Ižˆc


4(c)


J
K.lactisGG799
pKLAC1HSA
2Ižˆ
DNA
裧6mN
19kb
0=
,^ K.lactisGG799
Fá;µž€5[p

…ð
‡ˆSh
K.lactisGG799pKLAC1HSA
þ¤ªð

(a)M—DNAMarker1kb;2—

pKLAC1HSA;3—
F€>L

(b)M—DNAMarker1kb;2—
F€>L

(c)M—DNAMarker1kb;2
P
3—
Sh
K.lactisGG799pKLAC1HSA
2Ižˆ
PCR
>L

4—
Sh
K.lactisGG799pKLAC1
2Ižˆ
PCR
>L
;5—K.lactisGG799
Ižˆ
PCR

;6—PCR
J`-Û
f
4 
žÞ¸¡W^9Ù
Fig.4 Identificationofpositiveclones
23 YNB
Z„ë€^<=®
HSA
œ”^_`
  K.lactisGG799pKLAC1HSA
J|Ù2ÔK²
ݍ
YPG
r
YPG+134%YNB2
zGHIcGH

TÂ#$UÞ



›Þ

Pðñ0G


YNB
•–ˆ2Y¶Sì#?@

GHŸN
24h
ý
TÂco#
,^
7o´
YNB
•–ˆFŸN
36h
ýco#


YNB
éñ/,mæDS,´µ

A600²ÝP71P48。
f
5 K.lactisGG799pKLAC1HSA
„…ˆ‰
Fig.5 GrowthcurvesofK.lactisGG799pKLAC1HSA
  HSA

K.lactisGG799
c…2
SDS PAGE
ÖºyUÞ



›Þ



ìíGHIÊ
2&Â
,YNB
2o´PJðñ[\–”“rJK[
p

­GH
48h
201¯’©

J
HRP
Q=2ÿ
HSA
ÿ,d
Westernblot
û»

yUÞ



›Þ


:66×104
öNG|À`–è[p

ŸÏ
„
HSA
œ+ŸÏÞ,

Øð
HSA
Íą

+z

45×104
öá$„üý1˜
[8-9]
r(1˜
[10]
èë#…I€52JK[p

©
SDS PAGE
P
Westernblot
yPJäN


134%YNB
P
Jðñ[\01¯’©c–”“2NG

é7ð
ñBK…µ¶c
HSA
2JK

  
°±
K.lactisGG799pKLAC1HSA

YPG
r
YPG+134%YNB2
zGHIc…2y


0G
YNB
-1˜2TÂr
HSA
2…:ˆý/e
d2ç¤
。YNB
#ߪ²è
(NH4)2SO4、B8DT
Erü†s

sc
,(NH4)2SO4rü†sçè|H
85
T
 
L
 
´
 
Y
 
µ
 

  
!
10
"
 
Lp

{îéedTL(2çw
[11-12],
DéBKž
f{’6.,2Á2”“2JK
[13]。
DTErü
†sÏ*FD:F2_`

1˜ŽfgnyÔ
60"‡ß2ç¤
[14],
,m/1˜Ž2_`

[
\/Ž2YK>×

(^[\}”“LMý0
1©c

*{î[\/01¯’c2–”“

ËÀ
Ç#2²Ïƒ9

DPJST}”“FLMý0
1©c-Á2”“dJK
[15]。
f
6 
œ”ë
HSA
^
SDSPAGE
¹ºg¨
Fig.6 AnalysisofexpressiontendencyofproductHSA
1—Marker;2—K.lactisGG799pKLAC1HSA

YPG
cGH
48h¯
’©
;3—K.lactisGG799pKLAC1HSA

YPG+134%YNB
cGH2
48h¯
’©

4—K.lactisGG799pKLAC1

YPG
cGH
48h¯
’©
;5—K.lactisGG799pKLAC1

YPG+134%YNB
cGH2
48h¯
’©
f
7 
œ”ë
HSA
^
Westernblot

Fig.7 Identificationofexpressionproduct
HSAusingWesternblot
  
Á"

KLij2BKJK2pqD;
:①o
´”“F`¬

U
PMSF、EDTA
y
;②F¬ôõ[

89Úµ
、pH、
eàÔKy

J)”“F_å

③;¤”“F59~Sh;④-Á2”“d†„
y
[13、16-17]。
*pqÄY¶S2…céP[
\8‹KE
HSA
2JKD;7ÀdލÅÓ

24 
WXYÙÞg¨
  
Y¶S
K.lactisGG799pKLAC1HSA
è7GH
30
7

ÈÉ

7­Ùd
Westernblot
÷»(

yÚ
Þ
8。
›Þ



Œ7Sh2
HSA
…(è
81
μg/mL,67307Çè(77±03)μg/mL,݌ÆS
h…(2
94%,
!ðQY¶S2P6Y»`³

f
8 K.lactisGG799pKLAC1HSA
WXYÙÞg¨
Fig.8 GeneticstabilityanalysisofK.lactis
GG799pKLAC1HSA
3 
x
 
)
  
J•X–—D1˜èë#

ný²¤~…+
,
pKLAC1,
²Á2Iž
HSA
ðlý
pKLAC1
¯2
α MFtÝ。ž+,è9ØÁ2>Lã;Úº N
:

þ¤…+,Iný+,¯
Kex
”“F€»‚
A¯Ý2
XhoⅠ‚A。T¤+, pKLAC1— amdS


ž^PJˤ~Þ

{¥¦ˆmxqnv`
G9*

  
éÝ

+…Ú¡á$Þ»25A

U„
.…,ڀ°

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á±)

Oz

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YNB
aBK¯µö÷

y01
¯’©c2–”“ðñ[,
éJK[p>º$

  
^¯Tµ

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‰‘’“”“•X–—D1˜c2…
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HSA
…(è
81
μg/mL,HP6Y»。HŒÅÓ/ YNB-sT
Âr…234
,YNB
Péˆý/edŽTÂ

J)>×%

?@01IÊyç¤

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”“2LMrÁ2”“2JK

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L6j

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urements[J].JAmChemSoc,2004,126(43):1425814266.
[2] 
î;ã

†0

J‰‘’“”“è+,2Âx”“pQL2
ÅÓdh
[J].
c·MQTL®¯
,2008,3(6):454456.
[3] 
ÙÁ

%’<



•X–—D1˜…z{”“ÅÓdh
[J].
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