Development of cell lines stably expressing staphylococcal nuclease fused to dengue 2 virus capsid protein for CTVI.
To explore the potential application of capsid-targeted viral inactivation (CTVI) strategy in prophylactic model against dengue virus (DV) infection, here we fused a Ca(2+)-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 5mgr;/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.
Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China. qinede@sohu.com
Cheng-Feng
CF
Qin
E-De
ED
Qin
D000595:Amino Acid Sequence; D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D003716:Dengue Virus / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005814:Genes, Viral; D008836:Micrococcal Nuclease / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D038301:Virus Inactivation
D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; D008836:Micrococcal Nuclease