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[Co-localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and nucleolar phosphoprotein
Maojie Lv1, Jianfei Chen, Hongyan Shi
1Department of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China.
Objective:
To elucidate the co-localization characteristic between porcine epidemic diarrhea virus (PEDV) N protein and B23.1 phosphoprotein.
Methods:
Two pairs of primers used to amplify N gene and B23.1 gene were designed and synthesized according to CV777 N gene sequence (AF353511) and human nucleolar phosphoprotein B23.1 gene sequence (BC050628.1), respectively. The PEDV N gene and B23.1 gene were amplified by RT-PCR from PEDV strain CV777 and Vero E6 cells, respectively; then cloned into eukaryotic expression vector pAcGFP1-C1 and pDsRed2-N1, to generate the recombinant plasmids pAcGFP1-C1/N and pDsRed2-N1/B23.1, respectively. Vero E6 cells were transfected with plasmids pAcGFP1-C1/N and pDsRed2-N1/B23.1.
Results:
The fusion proteins successfully expressed in transfected Vero E6 cells by western blot analysis, and the PEDV N protein and the B23.1 phosphoprotein showed co-localization features in co-transfected cells through confocal microscopy analysis.
Conclusion:
The results will help to identify the nucleolar localization signals in PEDV N protein and to elucidate the mechanism of N protein located in nucleus.
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