點帶石斑魚 CD9 基因啟動子之活性與特性及添加 poly(I:C) 與感染石斑魚虹彩病毒的表現分析

(1)

Institute of Fisheries Science College of Life Science

National Taiwan University Master Thesis

CD9 poly(I:C)

Activity and Characterization of Orang-spotted Grouper (Epinephelus coioides) CD9 Promoter, and Its Expression

Analysis in Response to Poly(I:C) and GIV Infection

Chien-Wei Liu

Advisor: Chi-Yao Chang, Ph.D.

102 7

July 2013

(2)

(3)

(grouper Iridovirus, GIV)

(Epinephelus coioides) GIV

2012 poly(I:C) GIV LPS

poly(I:C) GIV CD9 (interferon, IFN)

IFN

CD9 2012 CD9 cDNA

CD9 GIV

CD9 cDNA

(Genome Walking) 1.2 Kb 2.6 Kb CD9 (promoter)

(transcription factor binding site) CD9

AP-1 ApoD C/EBP NF-κB TNF-α-Y-BOX IRF1 IRF2

(Luciferase Assay) GK (grouper kidney) GB

(grouper brain) 1.2 Kb CD9 2.6 Kb CD9

poly(I:C) GK CD9

1.2 Kb 2.6 Kb CD9 1.2

Kb CD9 124 bp 400 bp 679 bp

CD9 CD9 GK

poly(I:C) GIV CD9 NF-κB

c-rel GK CD9 RT-PCR

CD9 GK poly(I:C)

(Head Kidney, HK) (Spleen)

poly(I:C) RT-PCR CD9 HK Spleen

IFNa Mx

poly(I:C)

CD9 NF-κB

GIV CD9 GIV

(4)

Abstract

High economic benefits make groupers important aquacultural species in Taiwan.

However, the disease caused by grouper iridovirus (GIV) has resulted in economic losses due to high mortality in grouper culture.Therefore we take Epinephelus coioides which easily raised as our model to study the interactions between groupers and GIV. In 2012, Liu and Wu injected poly(I:C), GIV and LPS into orange-spotted groupers and discovered the expression pattern of CD9 resembling to those genes associated with interferon (IFN). IFN influencing antiviral effects are important bridges between innate immunity and adaptive immunity, but the role of CD9 remains unknown. We gained the total length of CD9 cDNA from orange-spotted grouper (Yang, 2012), finding that CD9 mainly expressed in immune organs. Thus, we hope to get CD9 promoter for further analysis to realize its relationship to GIV.

According to the total length of CD9 cDNA gained, we obtained 1.2 Kb and 2.6 Kb CD9 promoters from genome walking. Through analysis of sequence and transcription factor binding sites, we predicted that CD9 promoters might contain binding sites of AP-1, ApoD, C/EBP, NF-κB, TNF-α-Y-BOX, IRF1 and IRF2. By luciferase assay, we found that 1.2 Kb CD9 prmoter but not 2.6 Kb CD9 promoter regularly expressed in GK cells and GB cells, and their activities weren't affected by different concentration of poly(I:C). In deletion analysis of CD9 promoter in GK cells, 2.6 Kb CD9 promoter might have negative regulatory factor binding sites. Some binding sites located in 124 bp near the side close to transcription initiation point and the part between 400 bp and 679 bp of CD9 promoter might play a key role to affect expression. CD9 promoters with different lengths were also not affected by various concentration of poly(I:C), but the infection of GIV inhibit CD9 promoters, causing extremely low activity. Co- transfection with subunit of NF-κB, c-rel in GK cells up-regulated CD9 promoter.

(5)

Measuring endogenious CD9 expressions in response to poly(I:C) treatment in GK cells by RT-PCR showed no differences. Although the percoll purified white blood cells from head kidney and spleen of orange-spotted grouper showed no CD9 transcriptional difference after poly(I:C) treatment, IFNa and Mx expression showed significant up- regulation.

In summary, the CD9 transcriptional regulation might associate with NF-κB, and might be inhibited by the infection of GIV. However, the transcription factors involving in CD9 promoter regulation during GIV infection remain further study.

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... I ... II Abstract... III

... V ... VIII ... IX ... X

... 1

1.1 ... 1

1.2 (Iridovirus) ... 3

1.2.1 ... 3

1.2.2 ... 3

1.3 (tetraspanin superfamily) ... 4

1.3.1 ... 4

1.3.2 ... 5

1.3.3 ... 5

1.3.4 ... 5

1.4 CD9 (Cluster of Differentiation 9) ... 6

1.4.1 CD9... 6

1.4.2 CD9 ... 6

1.5 ... 7

... 8

...10

2.1 (cell culture) ...10

2.1.1 ...10

2.1.2 (passage) ...10

2.2 ...10

(7)

2.3 Genomic DNA ...10

2.4 (genome walking) ...11

2.5 (luciferase reporter vector) ...11

2.6 ...11

2.7 ...12

2.8 ... 12

2.9 ( luciferase assay)... 12

2.10 (titer) ... 13

2.11 Percoll ...13

2.12 RNA ...13

2.13 RT-PCR...14

...15

3.1 CD9 ...15

3.2 CD9 ...15

3.2.1 Poly(I:C) CD9 ...15

3.2.2 CD9 ...16

3.2.2.1 CD9 ...16

3.2.2.2 poly(I:C) GK CD9 ...16

3.2.2.3 GIV GK D9 ...17

3.2.2.4 c-rel CD9 ...17

3.3 RT-PCR...18

3.3.1 Poly(I:C) CD9 GK ...18

3.3.2 Poly(I:C) HK Spleen CD9 IFN Mx ...18

... 20

(8)

... 24

(9)

genome walking CD9 ... 32 CD9 1.2 Kb 2.6 Kb ... 33 1.2 Kb 2.6 Kb CD9

poly(I:C) CD9 ... 35

GK 2.6 Kb 1.2 Kb CD9 (deletion)... 36

GK poly(I:C) CD9 ... 37

GIV (MOI=10) CD9 GK

... 38

c-rel CD9 ... 39

Poly (I:C) CD9 GK ... 40

Poly (I:C) CD9 IFN Mx HK Spleen

... 41

(10)

... 43

(11)

pGL3... 45

pRL-CMV... 46

pcDNA3CF... 47

( ) ... 48

( ) ... 49

... 50

(12)

1.1

grouper Coral Trout

Rockcod (Serranidae) (Epinephelinae)

(Epinephelus)

10 ~ 30 1 ~

2 40 ~ 80

11 ~ 41 ‰ 15 ~ 35°C

400

110 (

) ( ) ( )

( ) ( ) ( )

(larva)

(fry)

(juvenile) (adult)

8~12

( ) ( )

(13)

(Protogynous hermaphrodite)

5

20 40

( ) 45 ~ 50

3~4 8~12

64

(14)

( 2007 2009)

1.2 (Iridovirus) 1.2.1

(International Committee on Taxonomy of Virus

ICTV) 2006 (Iridoviridae)

(Double-strand DNA virus) 120 ~ 200 nm

(Iridoviridae) Iris

(De Kinkelin and Bearzotti, 1981)

(Iridovirus

185 nm Chilo iridescent virus (CIV,

IIV6)) (Chloriridovirus 180 nm

Mosquito iridescent virus (IIV3, Invertebrate iridescent 3))

(Ranavirus 120 ~ 350 nm

Frog virus 3 (FV3)) (Lymphocystivirus 120 ~

350 nm Lymphocystis disease virus 1 (LCDV-1))

(Megalocytivirus 140 ~ 200 nm

ISKNV (infectious spleen and kidney necrosis virus)

1.2.2

(15)

1-2 (3.5-9.5 ) 100% 60%

25 5-30%

( 2003)

70

90

1.3 (tetraspanin superfamily)

1.3.1

(tetraspanins) 6

33

tetraspanins (integrins)

tetraspanins

tetraspanin (Monk and Patridge, 2012)

(16)

1.3.2

typeIII

(extracellular loop, EC) (EC1) (EC2) (Peter, 2012) (cysteine) palmitoylation ( ) site

Cys-Cys-Gly motif 2 6 cysteine

cysteine

(Seigneuret et al., 2001)

(Stipp, Kolesnikova, and Hemler, 2003)

1.3.3

(Stipp, Kolesnikova, and Hemler, 2003)

( Peñas et al., 2000)

1.3.4

( )

(Martin et al., 2005)

( )

CD81 HCV

(Hepatitis C Virus ) HCV E2

(17)

CD81 E2 HCV (de Knegt and van den Berg, 2001)

CD81-LEL Epstein-Barr virus B E2

(Pileri et al., 1998) CD82 antibody

HTLV-1 (Fukudome et al., 1992) CD63 HIV-

1 (Gluschankof et al., 1997) anti-

CD63 HIV-1 (von Lindern et al., 2003)

1.4 CD9 (Cluster of Differentiation 9) 1.4.1 CD9

CD9 (EC2)

Ser-Phe-Gln (Zhu et al, 2002) CD9

(Boucheix

and Rubinstein, 2001; Hemler, 2003) ( CD81

CD151) integrins EWI (EWI-2 EWI-F) (Hemler,

2005)

CD9 knockout

(Kaji et al., 2000; Le Naour et al., 2000; Miyado et al., 2000) CD9

CD9

1.4.2 CD9

CD9

HIV CD9 CD81 CD82 AP-3

(Garcia, Nikolic and Piguet, 2008) CD9/CD63

exosome (Wiley and Gummuluru, 2006) HeLa

HIV Gag budding site CD9 CD63 CD81

CD82 (Nydegger et al., 2006) Gag

CD9 (Krementsov et al., 2010)

CD9 CD81 CD63 Gag

(18)

(Krementsov et al., 2009, 2010; Rudnicka et al., 2009) HIV-1 CD9 CD81 CD82 CD231 (Meerloo et al., 1993)

36 CD9 CD81

(Shaw et al., 2008) CD9 K41 CD9

HIV (Khurana et al., 2007)

(feline immunodeficiency, FIV)

(Willett et al., 1994, 1995, 1997) FIV (de Parseval et al., 1997) (canine distemper virus, CDV) K41

(Löffler et al., 1997) (Schmid et al., 2000)

CD9 (adeno-associated virus-2,

AAV-2) (Kurzeder et al., 2007) CD9

1.5

GIV

2005 GIV

2011 poly(I:C) GIV

qRT-PCR 1 3 5 IFN

ISG15 DHX58 viperin CD9

poly(I:C) 1 GIV

CD9 IFN

" "

CD9 GIV

tetraspanin CD9 CD9

GIV

(19)

(1) Grouper Kidney (GK) cell (2) Grouper Brain (GB) cell (3) Head Kidney (HK) cell (4) Spleen (S) cell

(5) E. coli HIT-DH5 α strain (RBC bioscience) (6) E.coli XL1-Blue MRF’ strain (Stratagene) (7) E. coli TOP10F’ strain (Invitrogen) (8) T&A cloning vector (RBC bioscience) (9) pGL3 basic (Promega)

(10) pRL-CMV (Promega)

(11) pcDNA3CF (Yao and Yang, 2003) (12) GenomeWalker Universal Kit (Clontech) (13) Presto^TM mini Plasmid Mini Kit (Geneaid) (14) plasmid Midi kit (25) (QIAGEN)

(15) Dual-Luciferase^® Reporter Assay System (Promega) (16) HiScript I Reverse Transcriptase (BIONOVAS) (17) OligodT(20) primer (Invitrogen)

(18) EasyPure PCR/Gel Extraction Kit (Bioman) (19) Econo Taq 2X Master Mix (Lucigen) (20) PCR Pfu Master Mix II (GeneMark) (21) XhoI, KpnI (NEB)

(22) 50X TAE Buffer (BIOMAN) (23) T4 DNA ligase (Roche)

(24) Lipofectamine™ 2000 (Invitrogen) (25) Seakem® LE Agarose (LONZA) (26) Agar, Bacteriological (AMRESCO) (27) SDS (Bio-Rad)

(28) Ethidium bromide (EtBr, Merck) (stock 10 mg/ml) (29) SyBR safe

(30) TRIzol^® Reagent (Life Technology) (31) Phenol, saturated pH 6.6/7.9 (AMRESCO)

(20)

(32) Chloroform (MERCK) (33) Ethanol (MERCK) (34) 2-propanol (MERCK) (35) LB-Broth base (Invitrogen)

(36) 100 µg/ml Ampicillin sodium (MDBio, Inc) (37) Polyethyleneglycol 4000 (MERCK)

(38) PBS (Dulbecco’s Phosphate Buffer Saline) (39) FBS (Fetal Bovine Serum) (GIBCO) (40) Leibovitz’s L-15 medium (GIBCO) (41) Penicillin Streptomycin (GIBCO) (42) L-Glutamine 200mM (GIBCO)

(43) Dimethyl Sulfoxide (DMSO) (AMRESCO) (44) 0.5% Trypsin-EDTA (10X) (GIBCO) (45) 1 Kb Plus DNA Ladder (Invitrogen) (46) Sodium Chloride (BioShop)

(47) Diethyl pyrocarbonate (SIGMA) (48) Tris-HCl (MERCK)

(49) Percoll (Sigma)

(50) Cell Strainer 100 µm (BD Falcon) (51) DNA extraction buffer

10X DNA extraction buffer Tris-HCl (pH 8.0) 0.1 M

EDTA 0.01 M

NaCl 1 M

autoclave 1X DNA extraction buffer 10X DNA extraction buffer 100 µl

10% SDS 20 µl

10 mg/ml protease K 40 µl

dd H2O 840 µl

1000 µl

(21)

2.1 (cell culture) 2.1.1

(grouper kidney, GK) (grouper brain, GB)

10% FBS-L15 10% (Fetal Bovine Serum,

FBS) 100 units/ml penicillin 100 µg/ml streptomycin 2 mM L-glutamine

Leibovitz’s L-15 medium (GIBCO) 28 °C

2.1.2 (passage)

1X PBS (Dulbecco’s Phosphate

Buffer Saline) (GIBCO) 0.5% Trypsin-EDTA

5 ~ 7 10% FBS

trypsin

10% FBS 28 °C 3 ~ 4

2.2

350 L FRP

25 °C 30 ppt 12 1%

20

2.3 Genomic DNA

1 cm² 1X PBS

1.5 ml eppendorf 500 µl 1X DNA extraction buffer (

) 55 °C 1 10

eppendorf 1 (500 µl) pH 8.0 phenol

3, 000 rpm 10 tip

phenol

chloroform 3, 000 rpm 10

chloroform (1 ml) 100 % ethanol

(22)

tip genomic DNA 70 % ethanol 3, 000

rpm 5 5 dd H2O

DNA

2.4 (genome walking)

CD9 cDNA CD9-pro-sp2R

CD9-pro-sp1 ( ) genomic DNA Clontech

GenomeWalker Universal Kit Dra I EcoR V Pvu II Stu I 37 °C

overnight DNA phenol-chloroform

kit GenomeWalker Adaptor 16 °C ligation overnight

kit Adaptor Primer 1 (AP1) CD9-pro-sp2R PCR 94

°C 25 72 °C 3 7

94 °C 25 67 °C 3 32 67 °C 7

PCR Adaptor Primer 2 (AP2) CD9-pro-sp1 PCR

95 °C 5 DNA 95 °C 30 53 °C

30 72 °C 3 35 72 °C 7

TA cloning TFSearch TESS

Softberry NSITE

2.5 (luciferase reporter vector)

( ) PCR

CD9 PCR clean-up

pGL3 ligation kit

2.6

1.2 Kb 2.6 Kb CD9 PCR

CD9 pGL3 CD9

(23)

2.7

0.1 µg DNA

spin down 30 42 °C 2

(heat shock) 2 0.5 ml LB

37 °C 30 (3,000 rpm 5 )

LB 37 °C

2.8

Lipofectamine 2000 L15

5 ( Invitrogen Lipofectamine ^TM

2000 plate ) 20

L-

glutamine 20

2.9 ( luciferase assay)

Promega Dual-Luciferase® Reporter Assay System 1.5 × 10⁵ cells/ml GK 24-well plate overnight well 1 µl Invitrogen^TM lipofectamine 2000 0.7 µg pGL3 pGL3-CD9-1.2 K

pGL3-CD9-2.6 K GK 0.07 µg pRL-CMV

internal control 18 poly (I:C)

control 6 luciferase assay kit

1X PLB LARII 1X Stop & Glo reagent

PBS 100 µl 1X PLB shaker 15

(24)

PLB eppendorf 3, 000 rpm 5

20 µl 100 µl LARII 96 firefly

luciferase 10 1X Stop & Glo reagent renilla luciferase 10 firefly luciferase renilla luciferase

2.10 (titer)

1 × 10⁵/ml GK 0.5 ml 48

overnight

2 % FBS L-15 10^-1~ 10^-9 48

PBS

0.5 ml 28 °C (cytopathic effect, CPE)

7 Reed and Muench (1938)

2.11 Percoll

0.1

ppm 2-phenoxyethonl (HK) (S)

70% PBS 10% L15/FBS medium (BD)

4 ml 10% L15/FBS medium

4 ml 30% Percoll 4ml 50% Percoll 15 ml 4 °C 500 ×g

45 10% L15/FBS medium

1×10⁴ 6 cm

poly(I:C) 5 µg/ml RNA

2.12 RNA

(GK) 10% FBS-L15 6 cm (BD)

1 × 10⁶ 3 ml 10% L15/FBS overnight

PBS poly(I:C) 10% L15/FBS

RNA

PBS 1 ml TRIzol® (Invitrogen)

(25)

pipetman

1.5 ml 200 µl chloroform vortex 15~20

5 12, 000 ×g 15 ( 350 µl)

500 µl isopropanol 5

12,000 ×g 10 1 ml 70% DEPC-treated

RNA vortex 7,500 ×g 5

RNA 5 ~ 10 40 µl DEPC-

treated ddH2O

2.13 RT-PCR

RNA 0.5 µg RNA 1 µl

10 µM dNTP 50 pmole oligo dT(20) DEPC treated ddH2O

12 µl 65 °C 5 4 µl

5X First strand buffer 2 µl 0.1 M DTT HiScript I Reverse Transcriptase DEPC

treated ddH2O 8 µl 20 µl 42 °C 30 70

°C 15 cDNA 2 µl cDNA

1 µl 25 µl EconoTaq 2X Master Mix ddH2O 50 µl

94 °C 2 cDNA PCR 94

°C 30 CD9 β-actin IFNa 58 °C 30 Mx 53 °C 30

72 °C 30 35 72 °C 10 20

°C

(26)

3.1 CD9

CD9 IFN

cDNA 2 (genome walking)

CD9 1.2 Kb 2.6 Kb ( ) TA

cloning 1, 203 bp 2, 567 bp

22 bp CD9 1.2 Kb 2.6 Kb

TFsearch TESS Softberry NSITE 1.2

Kb 2.6 Kb IRF-1 IRF2 NF-κB TNF-α-Y-BOX

AP-1 AP-2 C/EBP ApoD ( ) IRF-1 IRF2 NF-κB

IFN

3.2 CD9

3.2.1 Poly(I:C) CD9

1.2 Kb 2.6 Kb CD9

1.2 Kb 2.6 Kb CD9 pGL3-CD9-1.2 K pGL3-CD9-2.6 K

pGL3-CD9-1.2 K pGL3-CD9-2.6 K GK 0.1 1 10 100 1,000 10,000 ng/ml poly (I:C)

pGL3 1.2 Kb 2.6 Kb

6 poly(I:C) ( A)

pGL3-CD9-1.2 K pGL3-CD9-2.6 K GB 1 10

100 1,000 10,000 ng/ml poly (I:C) pGL3

1.2 Kb 2.6 Kb 7

poly(I:C) ( B)

(27)

3.2.2 CD9

3.2.2.1 CD9

CD9 2.6 Kb 1.2 Kb 4 primer PCR

2, 208 1, 868 1, 529 1, 188 bp 1, 097 821 679 400 bp

CD9 1, 097 3' 5'

pGL3-CD9-2208 bp pGL3-CD9-1868 bp pGL3-CD9-1529 bp pGL3- CD9-1188 bp pGL3-CD9-1097 bp pGL3-CD9-821 bp pGL3-CD9-679 bp pGL3-CD9-400 bp

8 CD9 pGL3-CD9-1.2 K

pGL3-CD9-2.6 K GK 2.6 Kb CD9 2, 208 bp

2.6 Kb 7

1.2 Kb 1, 188 bp 1.2 Kb (

A) 2.6 Kb 1.2 Kb

1.2 Kb CD9 3' 124 bp 1097 bp CD9

1.2 Kb 31 % 5' 821 bp CD9

1.2 Kb 163.94 % 679 bp 1.2 Kb 136.92 % 400

bp CD9 1.2 Kb 12.04 % ( B) 400 bp

679 bp CD9

3.2.2.2 poly(I:C) GK CD9

CD9 GK 10

ng/ml 100 ng/ml poly(I:C)

( ) ( A) 2.6 Kb 1.2 Kb

( A) 1, 868 bp 2, 208 bp 1, 529 bp

(28)

3.2.2.3 GIV GK CD9

GIV CD9

CD9 GK GIV (MOI =

10) 9 GIV

1.2 Kb CD9 5% ( )

3.2.2.4 c-rel CD9

CD9 1.2 Kb 679 bp

NF-κB pGL3-CD9-1.2 K pGL3-CD9-679 bp

2000 c-rel

pcDNA3CF-c-rel ( 2010) GK CD9 NF-κB

CD9 (0.7 µg) 0.02 (0.014 µg) 0.04 (0.028 µg) 0.1 (0.07 µg) 0.2 (0.14 µg) c-rel

c-rel 1.2 Kb

1.2 Kb CD9 27% 679 bp CD9 c-rel

0.02 (0.014 µg) 679 bp CD9 34%

c-rel 0.02 (0.014 µg) 0.04

(0.028 µg) 0.2 (0.14 µg) 679 bp c-rel 0.2

( A)

c-rel CD9 (0.7 µg) 0.5

(0.35 µg) 1 2.5 (1.75 µg) 5 (3.5 µg ) pcDNA3CF-c-rel

GK 1 (0.7 µg) pcDNA3CF

pcDNA3CF 1.2 Kb CD9

0.5 (0.35 µg) c-rel pcDNA3CF c-rel

c-rel CD9 2.5 (1.75 µg)

pcDNA3CF 240% c-rel CD9 5 (3.5 µg)

2.5 (1.75 µg) 1 (0.7 µg) 2.5 (1.75 µg) c-rel

1.2 Kb CD9 c-rel

(29)

pcDNA3CF 679 bp CD9

c-rel 5 (3.5 µg )

c-rel c-rel

679 bp CD9 1.2 Kb c-rel (

B)

3.3 RT-PCR

3.3.1 Poly(I:C) CD9 GK

poly(I:C) CD9

poly(I:C) CD9 GK 100 ng/ml poly(I:C)

1 2 3 4 5 6 7 8 9 10 11 12 RNA RT-

PCR 100 ng/ml poly(I:C) CD9

( A)

GK 1 10 100 1, 000 10, 000 ng/ml poly(I:C)

5 hr 10 hr RNA RT-PCR

poly(I:C) CD9 ( B)

3.3.2 Poly(I:C) HK Spleen CD9 IFN

Mx

Percoll

5 µg/ml poly(I:C) 3 6 9 12 36

RNA RT-PCR poly(I:C) 5

48 HK

( A) Spleen

CD9 HK 5 µg/ml poly(I:C)

( B) Spleen ( C) IFN

(30)

HK poly(I:C) poly(I:C) 6

9 12 (

D) Spleen poly(I:C) 3 IFNa

( E) Mx HK poly(I:C)

6 ( F) Mx Spleen poly(I:C) 3

( G)

(31)

CD9 GIV IFN CD9

cDNA genome walking CD9

CD9 IRF1 IRF2 TNF-α-Y-

BOX NF-κB C/EBP AP-1 ApoD IRF1 IFN-α

IFN-β (Mamane et al., 1999; Taniguchi et al., 2001)

IFN-α IFN-β RNA IRF1 IFN-α IFN-β IFN-γ

IRF1 (Huang et al., 2009; Saha et al., 2010) IRF1

apoptosis (Gao et al., 2010) (tumor-suppression) (Cavalli et al.,

2010) IRF2 IRF1 IFN-α IFN-β

(Taniguchi et al., 2001) TNF-α

(Aroor et al., 2013) NF-κB

TNF-α

TNF-α (Napetschnig and Wu, 2013)

C/EBP CDK2

(cyclin dependent kinase 2) CDK4 kinases

(growth arrest) (Wang et al., 2001) AP-1 DNA

(Yu et al., 2013) ApoD apolipoprotein D apolipoprotein D (Do Carmo et al., 2002)

serralysin PAR-2 AP-1 C/EBP NF-κB

(Kida et al., 2007) Wang 2011 IPNV IRF1

IRF2 NF-κB C/EBPβ TNF-α

IPNV TNF-α/NF-κB

CD9 IFN TNF-α NF-κB

1.2 Kb CD9 2.6 Kb CD9 ( )

(32)

1.2 Kb 2.6 Kb CD9

CD9 2.6 Kb CD9

2.6 Kb CD9 1.2 Kb CD9 3' 124 bp

1.2 Kb CD9 31% 679 bp 1.2 Kb CD9 36 % 400 bp CD9

1.2 Kb 12 % ( ) 5' 400 bp ~ 679 bp

3' 1 ~ 124 bp CD9

3' 124 bp

IRF1 IRF2 400 bp ~ 679 bp TNF-α-Y-BOX

CD9 IRF1 IRF2 TNF-α

1.2 Kb CD9 2.6 Kb CD9 poly(I:C)

CD9 poly(I:C)

CD9 poly(I:C) ( ) GIV (MOI = 10)

GK CD9 GIV CD9

( ) GIV CD9 GK

" "

GIV GK " " CD9

CD9 NF-κB

IBDV

NF-κB apoptosis GIV CD9

GIV 9 GIV

MOI = 1 0.01 GIV

GIV CD9

CD9 NF-κB

NF-κB 1.2 Kb CD9 NF-κB 679 bp

CD9 2000 pcDNA3CF-c-rel GK

c-rel CD9 c-

rel 0.14 µg c-rel 1.2 Kb CD9

1.2 Kb CD9

(33)

(trans effect) ^R pcDNA3CF c-rel

0.7 µg c-rel c-rel

1.2 Kb CD9 1.75 µg

pcDNA3CF 2 679 bp CD9 c-rel 0.014 µg

3.5 µg 1.75 µg c-rel 679 bp CD9 ( )

c-rel 679 bp CD9 1.2 Kb CD9 679 bp CD9

c-rel 1.2 Kb 0.7 µg

c-rel 1.2 Kb CD9 679 bp CD9

NF-κB 679 bp CD9 c-rel

679 bp CD9 CD9

poly(I:C) CD9

GK GK CD9

RT-PCR poly (I:C) GK

CD9 CD9 GK

poly(I:C)

CD9 HK

48 Spleen 48

48 poly(I:C) HK Spleen

CD9 IFN Mx poly(I:C)

CD9 poly(I:C) IFN HK

6-9 12 36

Mx HK poly(I:C) 6

IFNa Mx IFNa HK

9 poly(I:C) HK IFNa

(34)

CD9 IFNa CD9

GK GB HK Spleen

CD9 CD9

CD9

poly(I:C) GK GB

HK Spleen

poly(I:C) GIV CD9 c-

rel GK 679 bp CD9 c-rel

1.2 Kb CD9 CD9 NF-κB

NF-κB 2011

poly(I:C) CD9 Zhang 2012

(pDCs) HCV

IFN-α

2011 CD9

2011

CD9 CD9

CD9

CD9 NF-κB GIV

CD9 NF-κB GIV CD9

(35)

(2003)

(2007) Handbook of Grouper (2009)

(2012) 2011 :

p. 37

(2010) 078R 030L

(2012) CD9

Aroor, A. R., McKarns, S., Demarco, V. G., Jia, G. and Sowers, J. R. (2013).

Maladaptive immune and inflammatory pathways lead to cardiovascular insulin resistance. Metabolism. pii: S0026-0495(13)00209-6.

Boucheix, C. and Rubinstein, E. (2001). Tetraspanins. Cell. Mol. Life Sci. 58, 1189- 1205.

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genome walking CD9

Clontech GenomeWalker^TM Universal Kit CD9

kit Dra I EcoR V Pvu II Stu I genomic DNA

DNA libraries kit adaptor PCR

Dra I Stu I libraries PCR 1.2 Kb 2.6 Kb M:

DNA size marker

1650 bp 2000 bp

850 bp 650 bp 500 bp 400 bp 300 bp 200 bp 100 bp 1000 bp 3000 bp

M Dra I Stu I

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0001 CCTGATACACACTGCTGAGGGAGATCTGCTGAAAAAACAGGTTTT 0045 0046 GAGCCAACGACCCAGCGTCAGTGTGAAGTGTCAGGACATCACTAA 0090 0091 CTACAGGAGACCATTCCCCCCCACTGTGGAGACCCAACAACTGGC 0135 0136 CGACGACCTGAATGTGTTTAACTGCAAGTTTAATGACCCCATCTT 0180

AP-1

0181 CACACCCCTCACCCACTCTGATTCAGACATCACATCCACACCTAA 0225 0226 CTCAGCCAGCTCACTGTCCCAGCCTCTACCTGTCAGTGGATCACA 0270 0271 CACAACTTCCTGACAGACCTGGAGCTGAACAGGCTCTAAACTATG 0315 0316 GAGATGACAATGGACTTCAGGATGAGCCCTCCATCACTGCCCCCC 0360 0361 TCACAATGCTGAACAACACTGTGATGTGGAATCCTGTAGGTAAAG 0405 0406 TGGACGTCCAACATCGACACCATCAGAAAAAAGGCCCAGCAGAGT 0450 0451 ACTTTCTGTGCCTGCTCAGGAGGTTCAGCCTCCCTTAGGAGCTGC 0495 0496 GGAGTATTTTAGAAACCGATGATCTACTAGGATTTTCACACACAA 0540 0541 CCATCTCTAGGGTTTACAGAGGATGGTCCCAAAATGTCTTGTTGA 0585 0586 TGCCAGAGGTCAGAGGAGAATAGCCAGACTGGTTCAAGATGATAG 0630 0631 AAAGGCAACAGGAAGTCAAATAACCACTGGTTACAACCAAGGTCT 0675 0676 GCAGAAGACCATCTCTGAAGCAACAACACCTTGTCCAACCTTGAA 0720 0721 GCAGATGGGCTACAGCAGCAGAAGACCACACCAGGTGCCACTCCT 0765 0766 GTCAGCTAACAACAGGAAACTGAGGCTACAGTTCACACAGGCTCA 0810 0811 CCAAAACTGGACAATAGAAGATTGGAAAAACCACATTCGGATGGA 0855 0856 AGCATGGATCCATCCTGCCTTGTATCAACGCTTCAGGCTGCTGCT 0900 0901 GGTGGTGTAATGGTGTGGGGGAGATTTTCTTAGTACCAACTGAGC 0945 0946 ATGGTCTAAACACCACAGCCTACCTGAGTATTGTTGCTGACCGTG 0990 0991 TCCATCCCTTTATGACCACAGTGTACCCATCTTCTGATGGCTACT 1035 1036 TCCAGCAGGATAACGCACCATGTCACAAAGCTCACATCATCTCAA 1080 1081 ACATGACAATGAGTTCACTGTACTCCAATGGCCTCCACAGTCACC 1125 1126 AGATCTCAGTCCAATAGAGCACCTTTGGGATGTGGTGGAACGGGA 1170 1171 GATTCTCATCATGGATGTGCAGCTGACAAATCTGCAGCAACTGTG 1215 1216 TGATGTCATCATGTCAATACGGACCAAAATCTCTGAGGAATGTTT 1260 1261 CCAGCGCCTTGTTGAATCTATGACACCAAGAATTAAGGCAGCTCT 1305 1306 GAAGGCAGAAGGGGTCCAGTGAGTGTATATAGTGATATATATATT 1350 1351 AAAAACAAAGAGCACTGTTAAGATGTTTAAACGTATTACCAGCTG 1395

AAACGTATTACCAGCTG AP-1

1396 CTCTATACTAAAAGATTAAACAGTTGTGTTTGTGTCAGTTGAAAG 1440 CTCAATACTAAAAGATTAAACAGTTGTGTTTGTGTCAGTTGAAAG 1441 GGATTTTTTTAGCATCTGGCCCCCAGGCATTTTCACATTATCTGT 1485

GGATTTTTTTAGCATCTGGCCCCCAGGCATTTTCACATTATCTGT 1486 TCTGGCCCCTGTTCAAAAACAGTCTGGACGCCCCTGATACAAACC 1530

TCTGGCCCCTGTTCAAAAACAGTCTGGACGCCCCTGATACAAACC 1531 AACAAACAACACCAGGAGTACAGACTTTGTACAAAGGTACTGCCT 1575

AACAAACAACACCAGGAGTACAGACTTTGTACAAAGGTACTGCCT 1576 CAGAGATACAACCAGAGCTAGAGAACGCTGTTGTCTGCTTTACAA 1620

CAGAGATACAACCAGAGCTAGAGAACGCTGTTGTCTGCTTTACAA ApoD(-)

1621 GGGTGCAATGCATCCTGTTGAATTGTGTATGTAGTTAATGTGTGT 1665 GGGTGCAATGCATCCTGTTGAATTGTGTATGTAGTTAATGTGTGT

ApoD(-)

1666 TTGTGTGAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG--- 1710 TTGTGTGAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG

ApoD(-) C/EBP NF-κB(-)

1711 ---CGGTGTAGCCTACGCATGAATGAGGTAAGAAGAGGGG 1755 TGTGTGTGCGGTGTAGCCTACGCATGAATGAGGTAAGAAGAGGGG

CD9 1.2 Kb 2.6 Kb

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1756 GAGGGACGGATAGCACATCACACTTCCTGTCCTTTGTTTCCTTTC 1800 GAGGGACGGATAGCACATCACACTTCCTGTCCTTTGTTTCCTTTC 1801 TTTGTTTTCAGCCCAAGTTTCACATTTGCCAAATCAAAACTGTCC 1845

TTTGTTTTCAGCCCAAGTTTCACATTTGCCGAATCAAAACTGTCC IRF1,IRF2 1846 AGTGCTCTTTTAATATAGTTCCTGCATGATAAATCTGTTCACTTC 1890

AGTGCTCTTTTAATATAGTTCCTGCATGATAAATCTGTTCACTTC 1891 AAACTGTTGGCTGCAGTGTGGGCTCCTGTTTGGCCTCATGTTTAT 1935

TNF-α-Y-box

AAACTGTTGGCTCCAGTGTGGGCTCCTGTTTGGCCCCATGTTTAT 1936 TTATGAGTTCCTGTGATTTTTATTACAACTCAAATCAGGTAGAAT 1980

TTATGAGTTCCTGTGATTTTTATTTCAACTCAAATCAGGTAGAAT 1981 AATAAAAAAAAAAGCCCGGACTAATCTCCAAAGCTGGTCCGTTTC 2025

AATAAAAAAAAA-GCCCGGACTAATCTCCAAAGCTGGTCCGTTTC AP-1

2026 CTGCTATCCACCTCATAAATAACTCATCCTTTATCATTCTTGACT 2070 CTGCTATCCACCTCATAAATAACTCATCCTTTATCATTCTTGACT 2071 CTCATTTCCTTAGGTTTCATTATGACTCTATGTTCTGCGGCCTCT 2115

CTCATTTCCTTAGGTTTCACTATGACTCTATGTTCTGCGGCCTCT 2116 TCGCGTCTGCGCGCCGCCGCCTGCTCATTGGTCCCACCTCGTGTC 2160

TCGCGTCTGCGCGCCGCCGCCTGCTCATTGGTCCCACCTCGTGTC 2161 TGTCACAGCTCTCAGCCAATGGAATGGCAGAGAGCGGTCAGAGAG 2205

TGTCACAGCTCTCAGCCAATGGAATGACAGAGAGCGGTCAGAGAG 2206 GCGGGGCATGACGCATGTCGGGACTGTATAACCAGCAGCTCAATG 2250

GCGGGGCATGACGCATGTCGGGACTGTATAACCAGCAGCTCAATG 2251 TGAAATCTGTGGATGTTCCACCACTGAAACTGCACGAGGACACAG 2295

TGAAATCTGTGGATGTTTCACCACTGAAACTGCACGAGGACACAG 2296 AGCGCGAGCGCCTTTGATCAACCAGGTATTTATGTAGTTTGAGGT 2340

AGCGCGAGCGCCTTTGATCAACCAGGTATTTATGTAGTTTGAGGT 2341 TAGTATCTGTAGCTCGGCTGCAACATGAATTAACTGCGCCATTGA 2385

TAGTATCTGTAGCTCGGCTGCAACATGAATTAACTGCGCCATTGA 2386 GATTTTTTAATGCGTTACTAACTGCTTAATACATTTACATGTCAA 2430

GATTTTTTAATGCGTTACTAACTGCTTAATACATTTACATGTCAA 2431 ATAAAACCTTTACAGGTTTGAGTATGTGTGAATGTTTTGATGTCG 2475

ATAAAACCTTTACAGGTTTGAGTATGTGTGAATGTTTTGATGTCG IRF1,IRF2

2476 CGTGCCGCAGACGGTCTGCACAAGTGATGATGTCATTTGTTTATT 2520 CGTGCCGCAGACGGTCTGCACAAGTGATGATGTCATTTGTTTATT 2521 GTGTACACATTTACAGATCTCGCAAACACACGCAGCACAACAACT 2565

GTGTACACATTTACAGATCTCGCAAACACACGCAGCACAACAACT 2566 CGGTGATCATTATGGC 2581 CGGTGATCATTATGGC

CD9 1.2 Kb 2.6 Kb

CD9 2.6 Kb 1.2 Kb CD9

CD9 TFSearch TESS NSITE

AP-1 AP- 2 ApoD(-) C/EBP NF-κB TNF-α-Y-BOX IRF1 IRF2

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A GK

B GB

1.2 Kb 2.6 Kb CD9 poly(I:C) CD9

0.7 µg pGL3 pGL3-CD9-1.2 K pGL3-CD9-2.6 K 0.07 µg pRL-

CMV internal control 18 poly(I:C) 6

firefly luciferase Renilla luciferase

± (A) CD9 GK poly(I:C)

0.1 1 10 100 1, 000 10, 000 ng/ml 1.2 Kb CD9

2.6 Kb poly (I:C) (B) CD9

GB 1 10 100 1, 000 10, 000 ng/ml

poly(I:C) poly (I:C)

0 5 10 15

pGL3 pGL3-CD9-1.2K pGL3-CD9-2.6K

Relative luciferase activity

0 ng/ml 0.1ng/ml 1 ng/ml 10 ng/ml 100 ng/ml 1000 ng/ml 10000ng/ml

0 5 10 15

pGL3 pGL3-CD9-1.2K pGL3-CD9-2.6K

Relative luciferase activity

0 ng 1 ng/ml 10 ng/ml 100 ng/ml 1000 ng/ml 10000 ng/ml

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A

B

GK 2.6 Kb 1.2 Kb CD9 (deletion)

1.2 Kb 2.6 Kb CD9 CD9

0.7 µg pGL3-CD9-2.6 K pGL3-CD9-2208 bp pGL3-CD9-1868 bp pGL3-CD9-1529 bp pGL3-CD9-1188 bp pGL3-CD9-1.2 K pGL3-CD9-1097 bp pGL3-CD9-821 bp pGL3-CD9-679 bp pGL3-CD9-400 bp pGL3 0.07 µg

pRL-CMV internal control GK 24 firefly

luciferase Renilla luciferase ±

1.2 Kb CD9 100% (A) 2.6 Kb CD9

1.2 Kb 1.2 Kb (B) 1.2 Kb CD9

3' 124 bp 1097 bp 31% 821 bp

679 bp 1.2 Kb CD9 400 bp

1.19

65.99 75.47 40.83

25.39 3.36

0.00 20.00 40.00 60.00 80.00 100.00 pGL3

1188 bp 1529 bp 1868 bp 2208 bp 2567 bp

Relative luciferase activity (%)

1.19 12.04

136.92

163.94 31.11

100.00

0.00 50.00 100.00 150.00 200.00 pGL3

400 bp 679 bp 821 bp 1097 bp 1203 bp

Relative luciferase activity (%)

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A

B

GK poly(I:C) CD9

0.7 µg pGL3-CD9-2.6 K pGL3-CD9-2208 bp pGL3-CD9-1868 bp pGL3-CD9-1529 bp pGL3-CD9-1188 bp pGL3-CD9-1.2 K pGL3-CD9-1097 bp

pGL3-CD9-821 bp pGL3-CD9-679 bp pGL3-CD9-400 bp pGL3 0.07 µg

pRL-CMV internal control GK 18 poly(I:C) 6

± 1.2 Kb 100% Poly(I:C)

0.00 20.00 40.00 60.00 80.00 100.00 pGL3

1188 bp 1529 bp 1868 bp 2208 bp 2567 bp

Relative luciferase activity (%) 0 ng/ml 10 ng/ml 100 ng/ml

0.59

53.34

173.69 198.93 59.39

100.00

0.00 50.00 100.00 150.00 200.00 250.00 pGL3

400 bp 679 bp 821 bp 1097 bp 1203 bp

Relative luciferase activtiy (%) 0 ng/ml 10 ng/ml 100 ng/ml

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A

B

GIV (MOI=10) CD9

GK

0.7 µg pGL3-CD9-2.6 K pGL3-CD9-2208 bp pGL3-CD9-1868 bp pGL3-CD9-1529 bp pGL3-CD9-1188 bp pGL3-CD9-1.2 K pGL3-CD9-1097 bp

pGL3-CD9-821 bp pGL3-CD9-679 bp pGL3-CD9-400 bp pGL3 0.07 µg

pRL-CMV internal control GK 18 GIV (MOI = 10)

9 firefly luciferase Renilla luciferase

± 1.2 Kb 100% GIV CD9

GIV CD9

0.97 1.58 2.15 1.83 1.76 0.22

0.97

107.14

139.25 110.51

73.77 10.60

0.00 50.00 100.00 150.00 200.00 pGL3

1188 bp 1529 bp 1868 bp 2208 bp 2567 bp

Relative luciferase activity (%) no treatment GIV:MOI=10

5.17 1.69

3.23 3.41 1.47 1.57

0.97

37.63

112.52 135.39 55.96

100.00

0.00 50.00 100.00 150.00 200.00

pGL3 400 bp 679 bp 821 bp 1097 bp 1203 bp

Relative luciferase activity (%) no treatment GIV:MOI=10

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A

B

c-rel CD9

0.7 µg pGL3-CD9-1.2 K pGL3-CD9-679 bp pGL3 pcDNA3CF 0.07

µg pRL-CMV internal control pcDNA3CF-c-rel GK 24

± 1.2 Kb CD9 100% (A) CD9

(0.7 µg) 0.02 (0.014 µg) 0.04 (0.028 µg) 0.1 (0.07 µg) 0.2

(0.14 µg) pcDNA3CF-c-rel (B) CD9 (0.7 µg) 0.5

(0.35 µg) 1 2.5 (1.75 µg) 5 (3.5 µg ) * (P < 0.05%)

0.00 5.00 10.00 15.00 20.00

pGL3 679 bp 1.2 Kb

Relative luciferase activity

0 pcDNA3CF 0.5:1 1:1 2.5:1 5:1 0.00 2.00 4.00 6.00 8.00 10.00 pGL3

679 bp 1.2 Kb

Relative luciferase activity

0 0.02:1 0.04:1 0.1:1 0.2 :1

* *

c-rel : CD9

* *

* * * * *

* *

*

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A

poly(I:C) : 100 ng/ml

CD9 β-actin

B

CD9 β-actin

poly (I:C) GK CD9

(A) GK seeding overnight 100 ng/ml poly(I:C) 1

2 3 4 5 6 7 8 9 10 11 12 RNA RT-PCR

poly(I:C) ctrl β-actin internal control CD9

100 ng/ml poly(I:C) (B) GK

seeding overnight 1 10 100 1, 000 10, 000 ng/ml poly(I:C)

5 10 RNA poly(I:C) ctrl β-actin

internal control poly(I:C)

1 2 3 4 5 6 7 8 9 10 11 12h ctrl

1 10 10

²

10

³

10

⁴

ctrl 1 10 10

²

10

³

10

⁴

ctrl

5 hr 10 hr

Poly (I:C) : ng/ml

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A

B HK

CD9

β-actin

C Spleen

CD9

β-actin

Poly (I:C) CD9 IFN Mx HK

Spleen

3 6 9 12 36 h Ctrl !!!!!!!!!!!!3 6 12 36 h Ctrl

HK S

S (polyIC:5h) HK (polyIC:5h)

HK (polyIC:48h)

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D HK

IFNa β-actin

F HK

Mx β-actin

E Spleen

IFNa β-actin

G Spleen

Mx β-actin

Poly (I:C) CD9 IFN Mx HK S

(A) Percoll HK Spleen 10% FBS L15

5 µg/ml 5 48 HK

( HK polyIC: 48h) Spleen

( ) (B) (C) (D) (E) (F) (G)

HK S poly(I:C) 5 µg/ml 3 6 9 12 36

RNA cDNA CD9 IFN Mx β-actin PCR

poly(I:C) (ctrl) β-actin internal control

!!!!!!!!!!!!!!3 6 9 12 36 h Ctrl

!!!!!!!!!!!!!!

3 6 12 36 h Ctrl

3 9 12 36 h Ctrl

!!!!!!!!!!!!!!

3 9 12 36 h Ctrl

(53)

CD9

(genome walking)

CD9-pro-SP2R 5’-TGCAGAAGAGGCCACATCCATCCA-3’

CD9-pro-SP1 5’-TGCCATAATGATCACCGAGTTG-3’

CD9

(promoter assay)

CD9-1203-F- Kpn1

5’-GGGGTACCCGTATTACCAGCTGCTCCATA C-3’

CD9-2567-F- KpnI

5’-GGGGTACCCCTGATACACACTGCTGAGGG AG-3’

CD9-1-R-XhoI 5’-CCGCTCGAGGATCACCGAGTTGTTGTGCT G-3’

CD9

(promoter deletion)

CD9-pro1.2F- 382

5’-GGGGTACCGACGGATAGCACATCACACTT C-3’

CD9-pro1.2F- 524

5’-GGGGTACCTCCAGTGTGGGCTCCTGTT T-3’

CD9-pro1.2F- 803

5’-GGGGTACCCAATGGAATGACAGAGAG CGG-3’

CD9-1203-F- Kpn1

5’-GGGGTACCCGTATTACCAGCTGCTCCATA C-3’

CD9-pro1.2R- 1079

5’-CCGCTCGAGCACGCGACATCAAAACAT TCAC-3’

CD9-pro2.6F- 359

5’-GGGGTACCCCTCACAATGCTGAACAAC ACTG-3’

CD9-pro2.6-F- 699

5’-GGGGTACCCAACACCTTGTCCAACCTT GAAG-3’

CD9-pro2.6-F- 1038

5’-GGGGTACCCAGCAGGATAACGCACCAT GTC-3’

CD9-pro2.6-F- 1379

5’-GGGGTACCAAACGTATTACCAGCTGC TC-3’

(54)

CD9

(RT-PCR)

CD9-mRNA-F 5’-ATGGCACTGGATGGATGTGGCC-3’

CD9-mRNA-R 5’-CACGGCGGAATAGTACGCAGTC-3’

Osg-β-actin-F 5’-GCCCCACCAGAGCGTAAATA-3’

Osg-β-actin-R 5’-CATCGTACTCCTGCTTGCTGAT-3’

IFN-F 5’-GTCCTTCCCGAATCATCTGTACAGCCA

G-3’

IFN-R 5’-GCTTGAGAGTCTCTTGAAGTACATGTG

CAGC-3’

Mx-F 5’-CTGATGGAGAGAGGACTCTG-3'

Mx-R 5’-CTGATGGCATCCTGAGTGAAAGC-3'

! !

(55)

pGL3

(56)

pRL-CMV

(57)

pcDNA3CF

(Yao and Yang, 2003)

(58)

( )

(Zöller, 2009)

(59)

( )

(Monk and Partridge, 2012)

(60)

(tetraspanin-enriched microdomain, TEM)

(Yáñez-Mó et al., 2009)