2.13 RT-PCR
3.3.2 Poly(I:C) HK Spleen
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
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)
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 ( )
1.2 Kb 2.6 Kb CD9
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)
NF-κB apoptosis GIV CD9
GIV 9
CD9 2000 pcDNA3CF-c-rel GK
c-rel CD9
c-rel 0.14 µg c-rel 1.2 Kb CD9
1.2 Kb CD9
(trans effect) R pcDNA3CF c-rel
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
CD9 IFNa CD9
GK GB HK Spleen
CD9 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
(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).
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genome walking CD9
Clontech GenomeWalkerTM 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
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
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
1 AP-2 ApoD(-) C/EBP NF-κB TNF-α-Y-BOX IRF1 IRF2
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)
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
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
Relative luciferase activity (%)
1.19
Relative luciferase activity (%)
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
firefly luciferase Renilla luciferase
± 1.2 Kb 100% Poly(I:C)
Relative luciferase activity (%) 0 ng/ml 10 ng/ml 100 ng/ml
Relative luciferase activtiy (%) 0 ng/ml 10 ng/ml 100 ng/ml
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
Relative luciferase activity (%) no treatment GIV:MOI=10
5.17
Relative luciferase activity (%) no treatment GIV:MOI=10
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
firefly luciferase Renilla luciferase
± 1.2 Kb CD9 100% (A) CD9
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
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
210
310
4ctrl 1 10 10
210
310
4ctrl
5 hr 10 hr
Poly (I:C) : ng/ml
A
B HK
CD9
β-actinC Spleen
CD9
β-actinPoly (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)
D HK
(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 Ctrl3 9 12 36 h Ctrl
!!!!!!!!!!!!!!
3 9 12 36 h CtrlCD9
(genome walking)
CD9-pro-SP2R 5’-TGCAGAAGAGGCCACATCCATCCA-3’
CD9-pro-SP1 5’-TGCCATAATGATCACCGAGTTG-3’
CD9
CD9-1-R-XhoI 5’-CCGCTCGAGGATCACCGAGTTGTTGTGCT G-3’
CD9-1-R-XhoI 5’-CCGCTCGAGGATCACCGAGTTGTTGTGCT G-3’
CD9-1-R-XhoI 5’-CCGCTCGAGGATCACCGAGTTGTTGTGCT G-3’
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'
! !
pGL3
pRL-CMV
pcDNA3CF
(Yao and Yang, 2003)
( )
(Zöller, 2009)
( )
(Monk and Partridge, 2012)
(tetraspanin-enriched microdomain, TEM)
(Yáñez-Mó et al., 2009)