Đánh giá sự biểu 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



 
và Interleukin--time PCR



i hc Khoa hc T nhiên
 ngành: ; 60 42 30

 2012

Abstract: -factor và geneMF(ALPHA)1; các protein
-
-time PCR; hóa 


CR;
-
             
-
time P--
-time PCR và Microarray.

Keywords: ; Gen mã hóa; ; 

Content
MỞ ĐẦU
        P. pastoris 
-


alpha (MF(ALPHA).
-
                  
(MF(ALPHA)1 
P. pastoris 

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 
Pichia pastoris 
Interleukin--



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CHƢƠNG 1: TỔNG QUAN TÀI LIỆU
1.1. Hệ thống biểu hiện Pichia pastoris
1.1.1. Đặc điểm hệ biểu hiện P. pastoris
P. pastoris   Saccharomycetaceae   
methanol.
          P. pastoris       
NRRL-Y 11430.
Bảng 1.1: Các chủng biểu hiện P. Pastoris
Tên chng
Kiu gen
Kiu hình
X-33
Chng di
Mut
+

GS115
his4
Mut
+
His
-

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

GS115/Albumin
his4
Mut
s

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

SMD1168

Mut

+
His
-
khuyt protease
SMD1165
Prb 1 his4
Mut
+
His
-
khuyt protease
1.1.2. Những ƣu điểm của hệ biểu hiện P. pastoris
P. pastoris   


 
nhanh chóng .
1.1.3. Quá trình chuyển hóa methanol ở P. pastoris
     

1.1.4. Cơ chế tiết protein ở nấm men

-S. cerevisiae 
1.1.5. Vector biểu hiện gene ngoại lai của P. pastoris
1.1.5.1. Đặc điểm chung của vector biểu hiện của P. pastoris




 

4
1.1.5.2. Vector pPIC9
1.1.6. Tích hợp gen ngoại lai vào P. pastoris
P. pastoris 
S. cerevisiae
P. pastoris 


+

s



+


+

-
+

1.2. Tổng quan về α-factor và gen MF(ALPHA)1

 MF1, MF2. Gen MF(ALPHA)1 P. pastoris 
     P. pastoris

1.3. Tổng quan về các protein ngoại lai trong đề tài
1.3.1. Tổng quan về Interleukin-2

Interleukin-2 (IL-


1.3.2. Tổng quan về α-amylase:

-S. fibuligera 

1.4. Kỹ thuật Real-time PCR
1.4.1. Tổng quan về Real-time PCR


-

--

-

cao.

5
1.4.2. Phân tích kết quả Real-time PCR
1.4.2.1. Biểu đồ khuếch đại của Real-time PCR (amplification graph)
1.4.2.2. Chu kỳ ngƣỡng (Ct)


1.4.2.3. Biểu đồ nóng chảy

  
 


càng


1.4.2.4. Phƣơng pháp so sánh định lƣợng tƣơng đối 2
ΔCt

-




Ct(C)-Ct(T)
.

6

CHƢƠNG 2: VẬT LIỆU VÀ PHƢƠNG PHÁP
2.1. Vật liệu
2.1.1. Chủng vi sinh vật và các gen biến nạp
P. pastoris .
- P. pastoris pPIC9 cng  control.
- P. pastoris ng Amy).
- P. pastoris pPIC9IL-2 ng IL-2).
2.1.2. Mồi cho phản ứng PCR và Real-time PCR
Tên mồi
Trình tự nucleotide
IPP1-Fw
-TACGGTGCCTTCCCTCAG-
IPP1-Rv
-TCCCTCATCCAACAAAGCCATAAC-

MF(ALPHA)1-Fw
-CTGCAGTTTTATTCGCAGCATCC-
MF(ALPHA)1-Rv1
-GCAGCAATGCTGGCAATAGTAG-
2.1.3. Hóa chất, enzyme và các thiết bị máy móc
 Hóa chất, enzyme:
 Máy móc và thiết bị:Máy Real-
2.1.4. Phần mềm

2.1.5. Các loại môi trƣờng và dung dịch
       
pastoris.

- 
và zymolase t
-                   

- 
-  


- 
- 

- 


7
- 



- 

-PAGE
2.2. Các phƣơng pháp nghiên cứu
2.2.1. Phƣơng pháp lên men và nuôi cấy các chủng P. pastoris tái tổ hợp
2.2.2. Phƣơng pháp tách chiết và tinh sạch mRNA từ P. Pastoris

- 

ông có RNase và DNase.
- 

- 

t mRNA tng s

600

 
5.10
7



7
0 vòng/ phút(v/p) 
0
C,
-ME and 250 unit


350 1.43
M - .  1
 70%, 











     i
700
ly tâm 1 ph 13000 13000

 13000v/ . 





u







, 







2 .



8
  

2.2.3. Kỹ thuật PCR
-



2.2.4 Kỹ thuật Real-time PCR
-time PCR nh





 


2.2.5. Xử lý số liệu sử dụng phần mềm LightCycler 4.0
Xác Real-time PCR,
i
là Ct , Ct 
u. Ct 

 Ct n ít chu 
có Ct 
        -Mono Color): Phân tích

 
   

ng Ct




      





9




10

CHƢƠNG 3: KẾT QUẢ VÀ THẢO LUẬN
3.1. Lên men các chủng P. pastoris tái tổ hợp
P. pastoris     
 P.
pastoris 

3.2. Tách chiết và tinh sạch RNA tổng số từ dịch nuôi cấy tế bào nấm men
3.2.1. Tách chiết RNA tổng số



3.2.2. Tinh sạch mRNA
           
-
[30]




 

- il-2.
- 
- -
không.




-



gene il-2 
Real-

11
                  
 Real-
time PCR hay không.

P. pastoris mang gene il-
P. pastoris mang gene mã hóa c

-

3.2.3. Đo hàm lƣợng các mẫu RNA tổng số
Mu
Ln lên men 1
Ln lên men 2
Ln lên men 3
ng/µL
A
260/280

ng/µL
A
260/28
0

ng/µL
A
260/28
0

A-0h
471,1
2
2,17
913,80
2,21
391,02
2,20
A-6h
619,3
7
2,24
620,53
2,21
385,47
2,20
A-
12h
324,9
7
2,20
614,46
2,21
326,78

2,22
A-24
585,0
5
2,23
142,87
2,20
507,42
2,23
A-
48h
672,9
8
2,21
449,67
2,18
617,99
2,22
A-
72h
521,0
2
2,19
1130,6
9
2,19
598,58
2,21
A-
96h

742,3
2
2,20
631,76
2,20
466,49
2,19
C-0h
399,8
8
2,18
802,78
2,21
457,88
2,15
C-6h
470,0
5
2,15
730,30
2,19
408,68
2,18
C-
12h
356,6
3
2,19
600,96
2,20

559,64
2,21
C-
24h
566,2
0
2,22
35,59
2,30
470,52
2,15
C-
48h
587,1
8
2,22
672,00
2,18
730,92
2,22
C-
72h
593,2
1
2,19
982,23
2,20
408,52
2,21
C-

96h
706,8
3
2,20
655,07
2,19
355,59
2,20
I-0h
581,6
5
2,23
717,58
2,21
361,24
2,18
I-6h
387,8
1
2,19
697,24
2,22
503,49
2,23
I-12h
320,9
2,20
604,04
2,22
776,55

2,22

12
6
I-24h
569,9
1
2,21
193,63
2,18
181,14
2,07
I-48h
686,0
0
2,23
1002,0
5
2,20
391,02
2,20
I-72h
704,1
5
2,21
1291,5
8
2,19
395,47
2,20

I-96h
916,1
5
2,22
656,81
2,19
336,78
2,22




-time PCR.
3.3. Tối ƣu điều kiện cho phản ứng Real-time PCR
3.3.1. Tối ƣu điều kiện Real-time PCR với gene chuẩn IPP1
 -time PCR

-
 -
-time PCR 


-
3.3.2. Tối ƣu phản ứng Real-time PCR với gene đích MF(ALPHA)1
3.4. Kết quả Real-time PCR với gene nội chuẩn IPP1

-
-

Biểu đồ khuếch đại gene IPP1 của các mẫu trong Real-time PCR.

Chu k ng (Ct)
Mu
Lên men ln I
Lên men ln II
Contro
0h
16.35
16.51

13
l

24 h
18.47
18.67
48 h
20.21
19.79

Amy

0h
15.96
16.55
24 h
18.64
18.51
48 h
20.43

19.65
IL-2

0 h
14.91
16.51
24 h
18.79
18.91
48 h
19.26
20.09
3.5. Real-time PCR về biểu hiện của gene MF(ALPHA)1
3.5.1. Kết quả Real-time PCR đoạn gene MF(ALPHA)1
MF(ALPHA)1 
-2 và amylase).
-
MF(ALPHA)1 
-
bên trên.
3.5.2. Phân tích so sánh mức độ biểu hiện gene MF(ALPHA)1
Thi
m thu
mu
Giá tr Ct trung bình
Control
Amy
IL-2
0h
25,60 ±

1,41
20,70 ± 0,26
20,02 ±
0,23
6h
26,77 ±
0,90
14,75 ± 0,48
16,26 ±
1,19
12h
26,56 ±
0,98
15,22 ± 0,47
15,61 ±
0,77
24h
26,40 ±
0,59
15,96 ± 0,78
16,84 ±
0,83
48h
26,52 ±
0,89
16,45 ± 0,31
17,01 ±
0,72
72h
26,51 ±

0,90
16,65 ± 0,35
17,19 ±
0,53
96h
26,72 ±
1,00
17,16 ± 0,35
17,64 ±
0,65
3.5.2.1. So sánh mức độ biểu hiện của gene theo thời gian lên men trong từng chủng.


So sánh
Control
0h
6h
12h
24h
48h
72h
6h
0,30
-
-
-
-
-
12h
0,34

1,16
-
-
-
-
24h
0,38
1,29
1,11
-
-
-
48h
0,35
1,19
1,03
0,92
-
-

14
72h
0,36
1,20
1,04
0,93
1,01
-
96h
0,31

1,04
0,90
0,80
0,87
0,86
Tỷ lệ khác biệt của gene MF(ALPHA)1 theo thời gian của cùng một chủng P.
pastoris mang gene mã hóa cho amylase theo giá trị chu kỳ ngƣỡng trung bình.
Thi gian
thu mu
Amy
0h
6h
12h
24h
48h
72h
6h
61,82
-
-
-
-
-
12h
44,74
0,72
-
-
-
-

24h
26,78
0,43
0,60
-
-
-
48h
19,03
0,31
0,43
0,71
-
-
72h
16,60
0,27
0,37
0,62
0,87
-
96h
11,67
0,19
0,26
0,44
0,61
1,42
Tỷ lệ khác biệt của gene MF(ALPHA)1 theo thời gian của cùng một chủng P.
pastoris mang gene mã hóa cho interleukin-2 theo giá trị chu kỳ ngƣỡng trung bình.

Thi gian
thu mu
IL-2
0h
6h
12h
24h
48h
72h
6h
13,55
-
-
-
-
-
12h
21,26
1,5
7
-
-
-
-
24h
9,04
0,6
7
0,43
-

-
-
48h
8,07
0,6
0
0,38
0,89
-
-
72h
7,11
0,5
2
0,33
0,79
0,88
-
96h
5,21
0,3
8
0,24
0,58
0,64
0,73
3.5.2.2. So sánh mức độ biểu hiện của gen MF(ALPHA)1 theo thông số về chủng.
3.6. So sánh kết quả Real-time PCR và Microarray
Phân tích Real-
MF(ALPHA)1   

-2.

15

KẾT LUẬN
-

-

-

    
Amylase và Interleukin-2.

KIẾN NGHỊ
 P. Pastoris 
sinh Amylase và Interleukin-        
ELISA.

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