Đánh giá sự biểu hiện Gen mã hóa Pheromone lai alpha (MF(ALPHA)1) ở các chủng Pichia pastoris tái tổ hợp sinh Amylase và Interleukin - 2 bằng kỹ thuật Real - ti

i













TRƢƠ
̀
NG ĐA
̣
I HO
̣
C KHOA HO
̣
C TƢ
̣
NHIÊN



THÂN VĂN MINH

ĐÁNH GIÁ SỰ BIỂU HIỆN GEN MÃ HÓA PHEROMONE LAI
ALPHA (MF(ALPHA)1) Ở CÁC CHỦNG Pichia pastoris TÁI TỔ
HỢP SINH AMYLASE VÀ INTERLEUKIN-2 BẰNG KỸ THUẬT
REAL-TIME PCR



LUÂ
̣
N VĂN THA
̣
C SI
̃
KHOA HO
̣
C








ii













TRƢƠ
̀
NG ĐA
̣
I HO
̣
C KHOA HO
̣
C TƢ
̣
NHIÊN



THÂN VĂN MINH

ĐÁNH GIÁ SỰ BIỂU HIỆN GEN MÃ HÓA PHEROMONE LAI
ALPHA (MF(ALPHA)1) Ở CÁC CHỦNG Pichia pastoris TÁI TỔ
HỢP SINH AMYLASE VÀ INTERLEUKIN-2 BẰNG KỸ THUẬT
REAL-TIME PCR


c thc nghim
: 60 42 30
LUC


NG DN KHOA HC
i

i  


i

LỜICẢM ƠN


inby tbitnsuscti:GS.TS.TrươngNamHải-Trng
phngKthutDitruyn,VintrngVinCngnghsinhhctntnhhng dn,
o mu ki .
Trongthigianhctpvmvic,tinhncsquantmgip, ch
botntnhvchuynmn,sngvinqubuca TS. Lê Thị Thu Hồng, TS. Dương
Cẩm Thúy,ThS.NguyễnHồng
Thanh, CN. Trần Nhật Anh

v  
tiphngKthutDitruynVinCngnghsinh hc,VinKhoa hc
CngnghVitNam.Nhndphonthnhlunvn ny,tixin bytlg
bitnsusccamnhti sgipvcgqubu.
Ticngxintrntrngcmnccthy gio,cgiotrongKhoaSinhhc-
TrngihcKhoa hcTnhin,ihcQucgiaHNi,nhgingdy, dudt
ti trongthigianhctptitrng.
Cuicg,tixingilicmnnngithntronggianhvbnb 
ngvinvgipti trongsutthigianhctpvnghincuvaqua.
Xin chânthànhcảmơn!

Ni,ngy12tng12m2012
Hcvin





ii

NHỮNG TỪVIẾTTẮT

Amp Ampicillin
Amy Amylase
AOX Alcochol oxidase
ARN RibonucleicAcid
DNA DeoxyribonucleicAcid
dNTPs Deoxyribonucleotidetriphosphates
EDTA Ethylenediaminetetraaceticacid
IL-2 Interleukin-2

kDa Kilodalton
LB Luria-Bertanimedium
MCS Multicloningsite
PCR PolymeraseChainReaction
PDB Proteindatabank
PDF ProbabilityDensityFunction
PMSF Phenylmethanesulfonylfluoride
Real-time PCR Real-time Polymerase chain reaction
SDS SodiumDodecylSulphate
TAE Tris-acetate-EDTA
TaqDNApolymerase DNAPolymerasecaThermusaquaticus
YNB Yeast Nitrogen Base


iii

MỤC LỤC
M U 1
U 3
1.1. H thng biu hin Pichia pastoris 3
m h biu hin P. pastoris 3
1.1.2. Nhm ca h biu hin P. pastoris 5
 P. pastoris 7
 tit protein  nm men 8
1.1.5. Vector biu hin gene ngoi lai ca P. pastoris 10
1.2. Tng quan v -eMF(ALPHA)1 12
1.3. Tng quan v   15
1.3.1. Tng quan v Interleukin-2 15
1.3.2. Tng quan v -amylase: 17
1.4. K thut Real-time PCR 18

1.4.1. Tng quan v Real-time PCR 18
t qu Real-time PCR 21
T LI 24
2.1. Vt liu 24
2.1.1. Chng vi sinh vn np 24
2.1.2. Mi cho phn -time PCR 24
t b  24
2.1.4. Phn mm 25
ch 25
u 27
ng P. pastoris  hp 27
ch mRNA t P. pastoris 27
2.2.3. K thut PCR 29
2.2.4. K thut Real-time PCR 30
2.2.5. X  liu s dng phn mm LightCycler 4.0 31


iv

T QU O LUN 33
ng P. pastoris hp 33
ch RNA tng s t dy t m men 36
t RNA tng s 36
3.2.2. Tinh sch mRNA 37
u RNA tng s 40
3.3. Tu kin cho phn ng Real-time PCR 42
3.3.1. Tu kin Real-time PCR vi gene chun IPP1 42
3.3.2. Tn ng Real-time PCR v 43
3.4. Kt qu Real-time PCR vi gene ni chun IPP1 44
3.5. Real-time PCR v biu hin ca gene MF(ALPHA)1 46

3.5.1. Kt qu Real-n gene MF(ALPHA)1 47
 biu hin gene MF(ALPHA)1 50
t qu Real- 53
KT LUN 55
U THAM KHO 56



v

MỤC LỤC BẢNG BIỂU

P. Pastoris 4
 41
-
P. pastoris 
 46
          -   
MF(ALPHA)1 P. pastoris  50
        MF(ALPHA)1      
P. pastoris  52
        MF(ALPHA)1      
P. pastoris 
 52
        MF(ALPHA)1      
P. pastoris -
 52

vi

MỤC LỤC HÌNH VẼ

P. pastoris 3
P. Pastoris 8
. 9
 11
 12
P. pastoris . 14
-2 16
-PCR 20


 22

 23
P. pastoris  33
P. Pastoris  34

P. Pastoris  35
 37
 38
 39
- 42
      -      
 43
IPP1 -time PCR. 45

vii

IPP1 trong Real-
time PCR. 45
MF(ALPHA)1 
trong real time PCR. 48
MF(ALPHA)1 
 49
l-
MF(ALPHA)1  49
eMF(ALPHA)1 
cP. Pastoris . 53

MF(ALPHA)1. 54


1

MỞ ĐẦU

 hc ng dng rc sn
xuu khoa hi protein mi
t biu hin ca mt h thng vt ch t c
u. Mt sn ph hp mi ph thuc rt nhi
vic chn vt ch biu hin, hin nay vt loi vt ch p cho
biu hin mi loc khi bu sn xut  m p
i la chn mt h thng biu hit nhiu h thng biu
hi biu hi h dng sinh v
  E.coli, Bacillus     m men Sacharomyces
cerevisiae, Pichia pastoris   

ng vt [3].
Nm men P. pastoris c s dng ph biu biu hin gen
ngon c biu hin trong P. pastoris. Vi
chng nu protein ngoc to ra vng l
t s protein lc biu hin  m thi 1
mg/l), hon  u hin ca protein, mt
s c s d  p, biu hin
ng thi vi chaperone hoc vt bin mt s gen ca
chng biu hin tu kii tt c ng
h  ng hiu bit v di truy  
trong chng nn ch.
Nhng kt qu  transcriptome ca chng P. pastoris i
chng, chng sinh tng hp interleukin-2, chng sinh tng hp amylase s dng
YEAST_2 array, 

 12.000 

















S. cerevisiae Schizosaccharomyces pombe cho the 
t trong biu hin gen giu  gene 
pheromone lai alpha (MF(ALPHA)1 [15].


2

GeneMF(ALPHA)1    -factor      n ca nm
men. Gene(MF(ALPHA)1   t trong chng P. pastoris g  u,
u hing nm men P. pastoris
o ch h
v m gia s biu hin ca gen ngoe(MF(ALPHA)1 
h biu hin.
Trong  biu hin ca gene(MF(ALPHA)1 ng
u s ng k thut Real-time PCR vi mc hi
 ng nht hic s d  kh nh li kt qu
microarray. K thut Real- biu hin ca gen s  nhy,
  thc hin vi s ng mu l 
biu hin ca gene(MF(ALPHA)1 t  ng cho nh
cu tip theo nh biu hin ca gen ngoi lai trong chng nm
men P. pastoris  hp.
c hi  biu biu hin gen
pheromone lai alpha (MF(ALPHA)1)  ng Pichia pastoris  hp
sinh a-2 bng k thut Real-
c ti thut di truyn, Vi Sinh
hc, Vin Khoa h Vit Nam.

3

CHƢƠNG 1: TỔNG QUAN TÀI LIỆU

1.1. Hệ thống biểu hiện Pichia pastoris
1.1.1.Đặc điểm hệ biểu hiệnP. pastoris
P. pastoris thuc h Saccharomycetaceae sinh v 
chuymethanol. Nt  elip
hon by chi n h
thc tip hp 



Tt c ng biu hin ca h biu hin P. pastoris n gc t
chng di NRRL-       ng di (X-33, Y-
      t bi  t histidinol dehydrogenase
t bie  dng
methanol (KM71, MC 100-ng khuyt protease (SMD1163, SMD1165,
SMD1168) [19].
Hình 1.1: Hình thái của nấm men P. pastoris[44]


4

Bảng 1.1: Các chủng biểu hiệnP. Pastoris
Tên chủng
Kiểu gen
Kiểu hình
X-33
Chng di

Mut
+

GS115
His4
Mut
+
His
-

KM71H
H
Mut
s
His
-

GS115/Albumin
His4
Mut
s

MC100-3
H

Mut
-
His
-

SMD1168

Mut
+
His
-
khuyt protease
SMD1165
Prb 1 his4
Mut
+
His
-
khuyt protease

Hu h t bin  genedehydrogenase histidinol (his4
a chu hin cha his4 khi bin np. khi DNA plasmid
c bin nng nhc ch hp 
ba ki  
+
, Mut
s
, Mut
-
[10, 12]. Ch    
+
 n tt
  ng ch  ng di (X-  ng b t
bin mt geneAOX1 t
s

ng b t bin mt c hai geneAOX

-
ng ch
 chng KM71H (his4 arg4 aox1Δ::AGR4) phn ln gen AOX1 b loi b 
bi geneAGR4 ca S. cerevisiaei dAOX2  to enzyme AOX
 ng trong methanol chm. Chng MC100-3 (his4 arg4 aox1Δ::
SAGR4 aox2Δ::Phis4    
-
, b loi b c hai gen AOX   bi
geneAGR4 genePhis4. Ch    
methanol. Tt c ng, ngay c cht bin Mut
-
   u
hin tt qua promoter AOX1. Mt s ch     
 n ch s a protease
mt yu t quan trm tit protein ngoc bi
 t  biu hin
i lai.


5

1.1.2. Những ƣu điểm của hệ biểu hiện P. pastoris
E. coli c s dt ch  biu hi hp t rt sm,
E. coli sinh v  c hin vic ci
bin sau dc biu hin b gii hc bi
ngun gc t sinh v n [18]. Biu hi    hp
trong E. coli ph thuc rt nhim ca protein mun biu hin.
Khi protein mun biu hin  dng tan nu vic to cc cao b li s dn ti

vic t p n 
 d n nhiu
thi gian, dn ti vic gim n sn phu hin. E. coli
   p cho nh  u biu hin protein c u ni
disulphide cao hoc nh      ci bin sau d  
o c
 hc to ra t E. coli  b i chng h
nhng h thng biu hiS. cerevisiae, P. pastoris
s dng[41].
i vi S. Cereviasiae c hin ci sau da
        c vi chi  i
carbonhydrate g t ln t 50-    
 cng th
  h  -  S.cerevisiae 
ch-1,3 glycan  u k
 protein [34,
37].
P. pastoris    c hin nhiu bi i sau d   
casinh vn   u (gm c d
gp cun, to cu n     
n gng   nh v
t c nh


6

 Pichia     ng vi s mannose ng  u so vi S.
cerevisiae-  P. pastoris ng ch gn chui
carbonhydrate vi 8- -1,3 glycan 
u k

[34]m ni trng h thng biu hin  
c [24]P. pastoris c la ch biu hi
ngun gc t sinh vP. pastoris  n vi
m u ln so vi S. cerevisiae, [10, 18, 24]  n
 di pH rng (t 3 t u ch t to protein,
gin phm ph m thiu tng ca protease [28].
Vi promoter m     ng protein to ra t P.
pastoris  n 30% tng s protein t [12]. Gene u khin bi
c kt h gen ca P. pastoris s tt chng
 u hin protein  mc cao v
c bi m ng bng methanol,
thun li trong vic kiu hin ca gene[4].
Cht cm ng biu hi tii cht cm ng
ca h thng biu hin E. coli n,
u t ng hot b sung
vt ti c thc hi
c   n xu 
  c ki
 thng biu hin bng phi tiy t 
t tin, ng mt nhiu tha hiu sut biu hin
protein l
n nay, P. pastoris c s  biu hi
hn gc t vi khun, nm, thc vng v
protein ci.


7

1.1.3. Quá trình chuyển hóa methanol ở P. pastoris
P. pastoris   dng

duy nh             
pero      c c     ng
chuy methanol trong ni mt h enzyme, mt trong s 
         n   
           chuy 
n o ra sn phm ph t
 c cho t       n    i xy ra trong
         thu h   i H
2
O
2
[9].
c chuyn t  
i ch 
AOX2. GeneAOX1 ch   a t 
u hin geneAOX1 t ch b c ch
ch cm  t cn thi 
geneAOX1  p vi
 m men tng hp mng l i [10,
12]t trong nh n trong qn h biu hin
P. pastorisu khin tng hc thit k 
tng hp protein ngoi lai.


8


1.1.4. Cơ chế tiết protein ở nấm men
Protein ngo c biu hin trong t  m men muc tit ra
khi t n phi cha mu tit    

  tit t a nm men c s dng nhi
SUC -- tip h[22,
39, 41]. M u tic s d
   u ti  -factor ca S. cerevisiae    
t [13].
u hin gene ngoi lai trong P. pastoris u tit
c thit k sc MF--u
n gc t nm men S. cerevisiaem khong 89 amino acid
 a mi ni cht d 
i ni ch tit MF- a protein s c loi b khi sn phm d
c tinh [9, 41]ng
thi ni cht protein ngoi qua s bi
c ch bin trong mi ni cht, protein
Hình 1.2: Con đƣờng chuyển hóa methanol trong nấm men P. pastoris[19]


9

s c vn chuyn th Golgi. T
dung hp v  ti[2].

C  c tng h  c d    
trong t t (1). phc hng hc
tng hc vn chuyi ni ch s hin din m
i ni ch2). Sau khi dt trong i ni ch
  n phc h Golgi nh  t (3), t  
ng h
ch i ni chc tng h
 c gi t (2a
Hình 1.3: Sơ đồ khái quát quá trình phân khu các protein ở sinh vật nhân

chuẩn[43]


10

  hp th b
ng: ti th 3a, 4a, lc lp 3b, peroxisome 3c, 4b ho
1.1.5. Vector biểu hiệngene ngoại lai củaP. pastoris
1.1.5.1. Đặc điểm chung của vector biểu hiện củaP. pastoris
u vector biu hiP. pastoris c thit k
yu t sau [19]:
1. Promoter mnh: t 
c biu hin mnh trong Pichia nh
 p v h gene nm men t   
 n k a gene AOX1 ng
p vi h gene nm men ti v [24].
t hoc nhiu v t ni (MCS): e mong mun
u hin.
3 t k n gc t geneAOX1 ca P. pastoris 
  
poly A  mRNA u qu cao.
4. Gene ch th chn lc (His4): n ca Pichia
histic s d b 
chng Pichia b t bin genehis4.
5. M cn thit cho s n ti ca plasmid trong vi khun
 e  chn lc t
 h hn ti
trong t n.
mt s vector biu hi 
 hc

phosphatase trong P. pastoris ho ca gene  gi
a S. cerevisiae[27].


11

1.1.5.2. Vector pPIC9
Khi s d biu hin protein ngo 
-D2, pPIC3.5, pHIL--D2, pPIC3.5
u hin protein n- biu hin
protein ngoc s dng nhiu trong P. pastoris.



    -948), v    -
u titor) (S) (949-1218), v -i
(1192-1241), v -n k
(1253-c m (ORF)HIS4 (4514--5626),
ColE1 origin (6708-n (713-6853)
1.1.5.3. Tích hợp gene ngoại lai vào P. pastoris
  u hin trong P. pastoris c thit k  ng vi locus
AOX1 hay locus his4. Gi S. cerevisiae, DNA dng thnh
n nP. pastoris nh  hp v 
ng trong b gene n h tn ti nh vi dn
sao trong b gene t .
Hình 1.4: Bản đồ vector biểu hiện pPIC9[42]


12

 chng Mut
+
hoc (Mut
s
), gene p ti locus his4 do s 
gia locus his4 ca nhim sc th nu hi5). Kt qu
 cho mt hoc nhiu bn sao ca vector ti v 
+
.
ng vector bng enzyme ct gii hn ti v 
+
hay MutS
ng ch s d hn sao ca
vector biu hin chim 1-ng His
+
bin np.




1.2. Tổng quan về α-factor và geneMF(ALPHA)1
S sinh sn  nm men S. cerevisiae n s ting c
peptide pheromone [26]  - 
ng v  ti  ng v alpha.
ng ca pheromone dn ti s dng li trong pha G1 ca chu k t 
i    bi cm ng ce 
  n ph             ng
pheromone. C t bin xuc pheromone s  sinh sn
c (s  t hp) [39].
 t chui peptide gi

2 gene ct MF1, MF2. Hai gene n peptide
(tin pheromone) lt g[39]. C hai ti t
Hình 1.5 Cơ chế trao đổi chéo tại vị trí his4 [25]


13

chuu, mu khi
u N), ti ng
vi mi tin nhn t. Tm ca geneMF1 ch lp li tt c
ng nht v cc nm men tit
m ca MF2 ch lp li, m  alpha
(Gln-5, Lus-- s t v
t alpha bi s thay th 2aa (Asn-5, Arg-     c g  -
[16]. Ti MF1 s tri qua nhic bi t
 c sn xut c-factor[27].
u v - alpha
trong vic cm ng nhing pheromone bao gm vic dng chu
k t m i ct 1 geneMF cho vic sn xut
  n, b t bin MF1/MF2 kh 
n xu  n [23]c b sung
 alpha cho chng MF1MF2 kh c kh n c
git bin. Nhng kt qu  c v
cht bie c alpha b , vic b 
t alpha t  c kh n ca th t
bin MF1/MF2 [41]. Mt gi thi git qu n
    nhi           n xut
    . Mt th t bi c bit ca mf1
(MF1 t hp   th
10 ln so vi th t biu (bt hot) MF1 mn b geneMF1[20, 22].

Th t bin MF1        u ca
geneMF1u ki m, theo sau bi rt nhiu amino acid
n URA3 [22, 39]. Kia th t bin MF1::URA3A
cho thy sn pha gene lai c ch n, MF
t bii ch u thuc geneMF1 ca ch


14

b ng s hp nhy geneMF(ALPHA) 
n ca nm nem S. cerevisiae[8].
GeneMFALPHA gn 2 gene eneMF(ALPHA)1 gene MF(ALPHA)2 
  -factor  t   n   n ln
c to ra bi geneMF(ALPHA)1.
 ceMF(ALPHA)1:
ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTC
CGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACA
AATTCCGGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGGGATTTC
GATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTT
TATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCT
TTGGATAAAAGAGAGGCTGAAGCTTGGCATTGGTTGCAACTAAAACCTG
GCCAACCAATGTACAAGAGAGAAGCCGAAGCTGAAGCTTGGCATTGGC
TGCAACTAAAGCCTGGCCAACCAATGTACAAAAGAGAAGCCGACGCTG
AAGCTTGGCATTGGCTGCAACTAAAGCCTGGCCAACCAATGTACAAAAG
AGAAGCCGACGCTGAAGCTTGGCATTGGTTGCAGTTAAAACCCGGCCAA
CCAATGTACTAA [44].
GeneMF(ALPHA)1    ng P. pastoris g  
trong vector biu hing nm men P. pastoris
o ch h

pPIC9
S
3‘TAOX1
HIS4
Amp
5‘PAOX1
HIS4
3’TAOX1 S 5’PAOX1 Amp 3’AOX1
his4*
Pichia genome
SMD1168 (his4)
* mutation
his4*
pPIC9-GOI
3‘TAOX1
HIS4
Amp
5‘PAOX1 S
GOI
HIS4
3’TAOX1 GOI S
5’PAOX1
Amp 3’AOX1
his4*
his4*
Pichia genome
SMD1168 (his4)
* mutation
A B

Hình 1.6: Thiết kế tạo các chủng nấm men P. pastoris tái tổ hợp[42]


15

A- chng control; B-     hp sinh protein ngoi lai: chng sinh
amylase hong sinh interleukin-2
AOX1
:  AOX1 promoter; S-
u tit -factor.(MF(ALPHA1));
AOX1
:  terminator; 
t  AOX1; Amp: gene p
a chng P. pastoris SMD1168 genome (AMY/IL-2).
1.3. Tổng quan về các protein ngoại lai trong đề tài
1.3.1. Tổng quan về Interleukin-2
Interleukin-2 (IL-2)  i c bi n d  u t 
ng t  ng min dch
mt ch tr u tr L-
mt cytokine do t a h thng min dch tng hL-c
ng s  sinh
ng c n gc t tL-t trong
nhnh  m . Geneil-2 
ng s  c
    ng s. V bn cht, IL-  t glycoprotein
c tii dng monome vi kh xp x 15 kDa. IL-2 tn ti vi
cu a cytokine loi 1.
IL-c gu t ng t  
m  -c quan
y nhiu kh u chnh min dc ca t 

 t t t t  t
(LAK). Tri sau d-2 b c
u ti -nh cha 133 amino acid.


16





t qu u cho th IL-t
nh hoc cc acid
amin 1-        c acid amin 121-   u
disulfide gi g [11]u ca Grace (1987) cho
tht bin mt 20 acid amin    t
99% hoa IL- t c th IL-
 acid amin cho thy IL-2 t a 3 gc Cys t
v o cu disulfide. Vi
 quyn hoc ca IL-
kt qu     t bin  gc Cy  y cu
 m 8-10 ln hoa IL- thay th hoc
t bia IL-2 gim ti 250 l t bin mt
gc Cys ti v  ng rn ho- i gc Cys
ti v ng ti hoa IL-2. Do vt bin thay
i Cys  v    n s t
mong mun gia gi hai gc Cys  v i u N ca
protein IL-c nhn bit b Ala-Pro-Thr  3 v 
Hình 1.7 : Cấu trúc không gian của IL-2 [40]