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ISSN
1672-9145
Acta Biochim Biophys Sin
2004, 36(8): 577–582
CN 31-1940/Q
Development of Cell Lines Stably Expressing Staphylococcal Nuclease
Fused to Dengue 2 Virus Capsid Protein for CTVI
Cheng-Feng QIN and E-De QIN*
Institute of Microbiology and Epidemiology, Academy of Military
Medical Sciences, Beijing 100071, China
Abstract To explore the potential
application of capsid-targeted viral inactivation (CTVI) strategy in prophylactic
model against dengue virus (DV) infection, here we fused a Ca2+-dependent nuclease, staphylococcal nuclease (SN), to the capsid
protein of dengue 2 virus (D2C) at the carboxyl terminal, and constructed the
desired expression plasmid pc/D2C-SN and control plasmids pc/D2C-SN* and
pc/D2C. A mammalian cell line BHK-21 was transfected by electroporation with
those plasmids and thereafter selected by 5 mg/ml blasticidin. The
resistant cell clones were then expanding cultured and screened by RT-PCR and
Western Blot assays. The nuclease activity of the expressed fusion protein
D2C-SN was analyzed by in vitro DNA digestion assay. It was confirmed
cell lines stably expressing D2C-SN and control constructs were obtained. The
intracellular expressed fusion protein D2C-SN had ideal nuclease activity and
no cytotoxicity on mammalian cells. Those engineered cell lines provided the
experimental system for CTVI application in prophylactic model and paved the
new road for combating DV infection with CTVI.
Key words dengue 2 virus; capsid
protein; staphylococcal nuclease; CTVI; stable expression
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Received: April 21, 2004
Accepted: June 19, 2004
This work was supported by a grant from the National Natural Science
Foundation of China (No. 30100006)
*Corresponding author: Tel, 86-10-66948614; Fax, 86-10-66948604;
E-mail, [email protected]