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

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


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

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

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
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 















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

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

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

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    

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

 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

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


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 

Trích đoạn Xử lý số liệu sử dụng phần mềm LightCycler 4.0 Tinh sạch mRNA Tối ƣu phản ứng Real-time PCR với gene đích MF(ALPHA)1 Kết quả Real-time PCR đoạn geneMF(ALPHA)1

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