Abstract: The eukaryotic expression plasmid of nonstructural protein Nsp1 of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) strain SX-1 was constructed, and the mutants of Nsp1 protein zinc finger 1 (ZF1) were obtained by site-directed mutagenesis, their expression activities in eukaryotic cells were examined. The Nsp1 target gene was amplified by RT-PCR from the total RNA of SX-1 strain, inserted into the eukaryotic expression plasmid of pCI-neo, and the positive plasmid was designated as pCI-neo-Nsp1. 8C, 10C, 25C and 28C in the Nsp1 protein ZF1 were mutated to A or S respectively by site-directed mutagenesis using pCI-neo-Nsp1 as template and 8 mutant plasmids were obtained. The plasmids were transfected into HeLa cells, the activity of Nsp1 protein was detected by indirect immunofluorescence assay (IFA) and Western blotting. The results showed that Nsp1 protein distributed in the cytoplasm and the nucleus using Flag antibody and Nsp1 protein mainly distributed in the nucleus using Myc antibody. Western blotting analysis showed S mutant proteins were about 44 and 20 ku and A mutant proteins were about 44 ku when detected by Flag antibody, while S mutant proteins were about 44 and 27 ku and A mutant proteins were about 44 ku when detected by Myc antibody. These results showed the eukaryotic expression plasmid of Nsp1 mutant were constructed successfully by site-directed mutagenesis and could be successfully expressed in HeLa cells, which provided the foundation for further researching whether ZF1 of Nsp1 protein downregulated the type Ⅰ IFN.

Key words: HP-PRRSV SX-1 strain; Nsp1; mutant construct; activity identification