Identification Of The Sub-cellular Localization Signal Of C Protein Of Classical Swine Fever Virus

Classical swine fever virus (CSFV) is the causative agent of disease in pigs. It is highlycontagious and deadly in pigs. Virulent strains can cause typical clinical symptoms such as highfever, neurological symptoms and bleeding,The prevalence of CSF for the pig industry caused alot of waste material and economic losses. Studies show that RNA virus will help to virusreplication through localizing nucleus in cells.So,the assay were test for CSFV C proteinsub-cellular localization characteristics and sub-cellular localization signal identification,which will help to understand the pathogenic mechanism of CSFV, and also can provide thebasis of new vaccines and anti-CSFV research strategy.In the study, we first design that Swine testicle cells were transfected with recombinantplasmids of pEGFP-CSFV-C expressing capsid protein and examined using confocalmicroscopy at24hours post transfection. The results revealed that the CSFV capisd proteinexpression mainly located in the nucleolus and cytoplasm in ST cells. In order to reduce thenuclear localization signal sequence,C gene was truncated into the two parts and the two partswere cloned into pEGFP-C2eukaryotic expression vector respectively,Then pEGFP-CSFV-C1,pEGFP-CSFV-C2recombinant plasmids were constructed. then swine testicle cells weretransfected pEGFP-CSFV-C1, pEGFP-CSFV-C2recombinant plasmids expressing proteinsalone. Confocal microscopy analysis the C expressing protein localization in swine testicle cellsafter24h.The results indicated that EGFP uniformLy localized to both the cytoplasm andnucleus, and pEGFP-CSFV-C1expressing protein localized to both the cytoplasm and thenucleus,the nucleolus not appear,wheras pEGFP-CSFV-C2expressing protein mainly localizedin the nucleus and cytoplasm,at the same time nucleolus were seen clearly.This data suggestedthat C2region may have a nucleolar localization signal. In order to accurately find the nuclearlocalization signal of C protein, C2is truncated again, and the truncated fragment to constructthe recombinant plasmid and corresponding to the same assay to transfect in swine testicle cells.The results show that the nuclear localization signal of C protein initially identified50VKKKGKVKGKNTQDGLYHNKNKP72in the C2region. The resuLts provide a basis forfurther identification of functional C protein nuclear localization signal.Research shows that CSFV C protein can localize to both the cytoplasm and nucleolus, thisfinding predicted that may C protein interact with nucleolar protein, and thus facilitate replication for the virus. To preliminary identify the interaction between CSFV C protein andNucleolin in swine testicle cells. Referring to sus scrofa Nucleolin gene in GenBank sequence,specific primers were designed and synthesized, the Nucleolin gene were amplified by PR-PCRfrom swine testicle cells. Then Nucleolin gene cloned into the eukaryotic expression vectorpDsRed2-N1,and constructed the pDsRed2-Ncu recombinant plasmid. Swine testicle cells wereco-transfected with pEGFP-CSFV-C and pDsRed2-Ncu recombinant plasmids expressingproteins and examined using confocal microscopy at24hours post transfection.pEGFP-CSFV-C and pDsRed2-Ncu occurred that the fusion proteins co-locatization features innucleolus. the result may be can provide a theoretical foundation for further exploring the CSFVC protein interaction with the Nucleolin protein.