Objective: To express and purify Porcine Reproductive and Respiratory Syndrome Virus Nsp2 protein and analyze the protease activity of Nsp2.
Methods: N-terminus and C-terminus of nsp2 gene were amplified by PCR and inserted into expression vector pET21a(+), respectively. The recombinant protein (Nsp2-N and Nsp2-C) were over expressed in E. coli BL21 and purified by Ni-NTA agarose affinity chromatogram and gel filtration. There is a cysteine protease domain (CP) in Nsp2-N through genetic alignment. In this study, the protease activity of Nsp2-N in cis was analyzed by western blot. Additionally, the predicted peptide substrate were synthesized, the protease activity in trans was analyzed by peptide cleavage assay in vitro.
Results: Two soluble recombinant protein were successfully expressed and purity reached up to 90% after purification. The putative protease domain of Nsp2-N couldn't cleave the predicted substrate through cis cleavage and trans cleavage.
Conclusion: The putative protease domain in Nsp2-N may need other host factors to act as cofactor, which supply basis for further identification of biological activity and screening of anti-virus drug.