Method for Japanese encephalitis virus NS3 protease activity analysis and high-throughput screening assay for inhibitors / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 194-202, 2014.
Artigo
em Chinês
| WPRIM
| ID: wpr-279530
ABSTRACT
Japanese encephalitis virus (JEV) is a single-stranded and positive-sense RNA, which has a single ORF (open reading frame), encoding a polyprotein precursor. Non-structural protein 3 (NS3) plays an important role in processing the polyprotein precursor and has become an important drug target of flavivirus. In this study, NS2BH-NS3 gene was amplified by PCR and subcloned to the prokaryotic expression plasmid, resulting pET30a-NS2BH-NS3. The fusion protein was expressed in Escherichia coli BL21 (DE3) in soluble form after induction by Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). The recombinant protein was purified by Ni-NTA affinity column. Then a fluorescence resonance energy transfer (FRET) method was used to determine enzymatic activity and the assay conditions were optimized. After screening 113 compounds, we found two compounds inhibiting the activity of NS2BH-NS3. This study provides a convenient and cost-effective method for screening of JEV NS3 protease inhibitor.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Inibidores de Proteases
/
Proteínas Recombinantes de Fusão
/
Serina Endopeptidases
/
Química
/
Proteínas não Estruturais Virais
/
RNA Helicases
/
Vírus da Encefalite Japonesa (Espécie)
/
Escherichia coli
/
Ensaios de Triagem em Larga Escala
/
Metabolismo
Tipo de estudo:
Estudo diagnóstico
/
Estudo de rastreamento
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2014
Tipo de documento:
Artigo
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