My Summer

~~ 在風裡的男人在風裡的男人 ~~ 891607林明興

中研院 分生所

Dr. Huey-Nan Wu 吳惠南

研究方向

D 型肝炎病毒抗原核酸監護子活性的研究

登革熱病毒複製機制的研究

Introduction (I)Dengue Virus

C prM E NS1 NS2 NS3 NS4 NS5

S.S. RNA

Structureprotein

Non-structure protein

NS3 N-terminal Helicase

NS3 C-terminal Protease

NS5 N-terminal RdRp

NS5 C-terminal Methylase

Introduction (II)

RNA cDNA

Clone

Expression

Purification

My Project

Clone, expression and purification

for DENGUE virus type II & III

NS5 N-terminal domain Protein

Clone for Dengue NS5-N

Amplify cDNARecombine to vector, pET21bTransform to competent cell, JM101Checking with restriction enzyme

Expression

Recombinant DNA transform to competent cell, BL21DE3Cell cultureAdding IPTG to induce protein expressionSDS-PAGE analysis

Growth Curve

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time course (hr)

OD

600

nm A 325II5N-11

B 325II5N-17

C 325II5N-18

D 325II5N-20

E 325II5N-21

DNII5N-11 positive control

Protein purification

1. Reagent preparation2. Cell extract preparation3. Resin preparation (charge with

Ni+)4. Cell extract flow through column5. Eluting the target protein

Small scale purification – batch method

Purifying with various salt concentration

Eluting without denature reagent, urea

Eluting with lower concentration imidazole

Various NaCl concentration

Urea & Imidazole

Imidazole

Large-amount purification – column chromatography

Large-amount purification – column chromatography

Some memories

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