Summer Research Inhibition of Type I Interferon Signal Transduction

Pathways by JEV infection

中央研究院生物醫學科學研究所林宜玲 老師

University of California, Los Angeles

Summer Student: 陳勃睿 清華大學生科 05 級2003/12/5

Introduction

Flaviviruses

Journal of Infection (2001) 42, 104–115

Introduction

Flaviviruses Enveloped virus Single-strand, positive sense RNA as genome One ORF encoding a polyprotein

CprM

/M E NS1NS1

NS2ANS2BNS3

NS4ANS4B NS5

: host signalase: viral protease NS2B-3: furin or other golgi-localized protease: unknown protease

* *

*: glycosylation

Introduction

Interferons (IFNs) are a family of cytokines that procee

d antiviral activity.

-Type I (viral IFNs) include IFN-α, IFN-β, IFN-ω, IFN-τ

produced by most body cells

-Type II (immune IFNs) include IFN-γ

produced by certain immune cells only such as CD4+ T help c

ells

Introduction

Signal Transduction Pathways

Introduction

Viral interference in the IFN system

Material

Japanese Encephalitis Virus

- E domain II, prM/M, EpIRES-hrGFP, r21-TE12QCompetent Cells: Top10, DH5αTransfection: BHK-21 (mouse)

Method

Molecular Cloning TechniqueTransfectionImmunofluorescence Assay (IFA)Western BlottingPolymerse Chain Reaction (PCR)Deletion with one primer in single step

Molecular Cloning

JE-II/pSecTag B

Nhe I

T4 DNA polymerase

Sal I

養菌，抽 plasmid

Gel Extraction

pIRES-hrGFP

Sma I

Sal I

CIP (AP)

Ligation

Transformation

Transfection

Lipofectamineartificial membrane

Sindbis Virus interferon-sensitive virus

Immunofluorescence Assay

DAPI

Immunofluorescence Assay

DAPI

Western Blotting

Failed...

Single Primer Deletion

higher efficiency than primer pairs

(although less colonies)DpnI (5’-Gm6ATA-3’)cost is reduced

BioTechniques Vol.29, No.5, 970-972

Lab Members

Thank you!