My Summer ~ 在風裡的男人 ~ 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 Structure protein Non-structure protein NS3 N-terminal Helicase NS3 C-terminal Protease NS5 N-terminal RdRp NS5 C-terminal Methylase

Introduction (II) RNAcDNAClone ExpressionPurification

My Project Clone, expression and purification for DENGUE virus type II & III NS5 N-terminal domain Protein

Clone for Dengue NS5-N Amplify cDNA Recombine to vector, pET21b Transform to competent cell, JM101 Checking with restriction enzyme

Expression Recombinant DNA transform to competent cell, BL21DE3 Cell culture Adding IPTG to induce protein expression SDS-PAGE analysis

DNII5N-11 positive control

Protein purification 1. Reagent preparation 2. Cell extract preparation 3. Resin preparation (charge with Ni + ) 4. Cell extract flow through column 5. 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

Some memories

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