The prokaryotic expression, polyclonal antibody preparation, and subcellular localization of the transmembrane protein NS2A of the duck Tembusu virus

Abstract

Duck Tembusu virus (DTMUV) is a single-stranded, positive-sense RNA arbovirus, belonging to the genus Flavivirus, the family Flaviviridae. As a transmembrane protein, non-structural protein 2A (NS2A) plays an important role in virion assembly, replication complex and antagonizing host immune response. Since NS2A protein contains many hydrophobicaminoacids, it is hard to gain the full-length protein of NS2A for prokaryotic expression. Therefore, to make a deep study, prokaryotic expression and polyclonal antibody preparation of truncated DTMUV NS2A was performed. The truncated NS2A gene (178-450 bp) was obtained, and sub-cloned into the prokaryotic vector pGEX-4T-1 (pGEX-4T-1-NS2A178-450bp). Subsequently, the recombinant GST-NS2A60-150aa protein was successfully expressed in E. coli BL21 (DE3) with the induction by 0.3 mmol/l isopropyl β-D-thiogalactoside (IPTG) for 6 h at 37°C. The GST-NS2A60-150aa protein was extracted from the gel. The BALB/c mice were immunized with the purified recombinant NS2A protein to prepare polyclonal antibodies against the truncated NS2A protein. The titer of the polyclonal antibodies, determined by ELISA analysis, was 1:128,000. The specificity of the polyclonal antibodies (mPAb-DTMUV-NS2A60-150aa) were verified by Western blot analysis. Furthermore, the indirect immunofluorescence (IFA) was performed to explore the subcellular localization of NS2A. NS2A protein was, in the transfected cells, located mainly around nucleus in the endoplasmatic reticulum. Taken together, our study provided a useful tool for the further exploration of the biological functions and molecular mechanism of DTMUV NS2A. Keywords: duck Tembusu virus; non-structural protein 2A; prokaryotic expression; polyclonal antibodies; subcellular location.