Download viral protease NS2B-3

Summer Research
Inhibition of Type I Interferon Signal Transduction Pathways by
JEV infection
中研院生醫所 R443
林宜玲老師
清大生科 05 蔡文揚
2003
實驗室簡介
• 研究Flavivirus-日本腦炎病毒 登革熱病毒
• 黃質病毒的基因體為單股正向的RNA，能夠作出一個
大蛋白質，經過切割後形成大約11個病毒結構性以及
非結構性蛋白質。
* *
: host signalase
: viral protease NS2B-3
: furin or other golgi-localized protease
: unknown protease
Interferons (IFNs) are a family of cytokines that possess antiviral
activity.
Type I IFNs (viral IFNs)
include IFN-a, IFN-b, IFN-w and IFN-t
produced by most types of cell can produce
Type II IFN (immune IFN)
IFN-g
produced only by NK cells, CD4+ T help cells and CD8+ cytotoxicity cells
IFNs-mediated signaling pathway and anti-viral strategies
Viral interference with the IFN system
Our works
• Construct JEdomainE/pIRES-hrGFP
• Construct prMM/ME/pR21-TE12Q
Construct JEIII/pIRES-hrGFP
ATG
Myc epitope
pSecTagA pCR3.1 JEV-E III pCR3.1 pSecTagA
BamH1
Kpn1
Asp7181
Xho1
Xba1
Hind III
Sfi1
Apa1
Nhe1
Sal1
Sma1
Sal1
pIRES
hrGFP
JEV-E III/pSecTagA
IRES-hrGFP
Sma1(blunt
end)
Nhe1
T4 DNA polymerase
Clean up
Clean up
Sal1
Sal1
Gel elution
Clean up
Ligase
Ligation
Transformation
CIP
Transfection
• Lipofectamine (artificial membrane)
• BHK-21
Immunofluorescence Assay
Western plot
IFA
IRES-hrGFP
control
DAPI
Myc+Texas Red
JE-III/pIRES-hrGFP
DAPI
Myc+Texas Red
Construct prMM/ME/pr21TE12Q
• Fail………….
心得
• 科學家一定要心地善良