Establishment and identification of classical swine fever virus (CSFV) capsid targeted nuclease expression system.
- Author:
Bin ZHOU
1
;
Ke LIU
;
Pu-Yan CHEN
Author Information
1. Key Lab of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China. aidzhou77@yahoo.com.cn
- Publication Type:Journal Article
- MeSH:
Animals;
Capsid Proteins;
genetics;
metabolism;
Cell Line;
Classical Swine Fever;
virology;
Classical swine fever virus;
genetics;
metabolism;
Gene Expression;
Genetic Engineering;
Micrococcal Nuclease;
genetics;
metabolism;
Plasmids;
genetics;
Recombinant Fusion Proteins;
genetics;
metabolism;
Swine
- From:
Chinese Journal of Virology
2008;24(6):451-455
- CountryChina
- Language:Chinese
-
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.
