Development of a monoclonal antibody against canine parvovirus NS1 protein and investigation of NS1 dynamics and localization in CPV-infected cells

Abstract

Canine parvovirus (CPV) non-structural protein-1 (NS1) plays crucial roles in CPV replication and transcription, as well as pathogenic effects to the host. However, the mechanism was not fully understood. Lack of NS1 antibody is one of the restricting factors for NS1 function investigation. To prepare NS1 monoclonal antibody (mAb), the NS1 epitope (AA461 ~ AA650) gene was amplified by PCR, and inserted into pGEX-4T-1vector to construct the prokaryotic expression vector of GST-tag-fused NS1 epitope gene. The NS1 fusion protein was expressed in E. coli, and purified with GSH-magnetic beads, and then used to immunize BALB/c mice. The mouse splenic lymphocytes were isolated and fused with myeloma cells (SP 2/0) to generate hybridoma cells. After several rounds of screening by ELISA, a hybridoma cell clone (1B8) stably expressing NS1 mAb was developed. A large amount of NS1 mAb was prepared from mouse ascites fluid. The isotype of NS1 mAb was identified as IgG1, which can specifically bind NS1 protein in either CPV-infected cells or NS1 vector-transfected cells, indicating the NS1 mAb is effective in detecting NS1 protein. Meanwhile, we used the NS1 mAb to investigate NS1 dynamic changes by qRT-PCR and location by confocal imaging in CPV-infected host cells and showed that NS1 began to appear in the cells at 12 h after CPV infection and reached the highest level at 42 h, NS1 protein was mainly located in nucleus of the cells. This study provided a necessary condition for further investigation on molecular mechanism of NS1 function and pathogenicity.