Establishment and identification of classical swine fever virus (CSFV) capsid targeted nuclease expression system / 病毒学报

ABSTRACT

One pair of primers was designed based on the sequence encoding capsid protein C of classical swine fever virus (CSFV). The C gene fragment was amplified by RT-PCR and PCR products were inserted into eukaryotic expression vector pcDNA-SN containing staphylococcal nuclease (SN) gene resulting in recombinant plasmid pcDNA-C-SN. 48h after transfection of the recombinant into porcine kidney (PK)-15 cells using liposome, the expression of fusion protein was identified through RT-PCR, Western blot and indirect immunofluorescence, and nuclease activity was detected by in vitro DNA digestion assay. The results showed that fusion protein of C-SN was expressed stably in PK-15 cells, and could be identified by rabbit polyclonal antibody against CSFV capsid protein and had good nuclease activity to cleave DNA. Meanwhile, the expressed fusion protein of C-SN in the transfected cells could effectively inhibit the proliferation of CSFV, reducing the infection rate by 10(2)-10(3) times. Our findings laid a foundation for further application of capsid-targeted antiviral strategies for CSFV.