[Establishment and identification of classical swine fever virus (CSFV) capsid targeted nuclease expression system].
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.
Zhou
Bin
B
Key Lab of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China. aidzhou77@yahoo.com.cn
Liu
Ke
K
Chen
Pu-Yan
PY
D000818:Animals; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D006691:Classical Swine Fever / Q000821:virology*; D006692:Classical Swine Fever Virus / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine
D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; D008836:Micrococcal Nuclease