Characterization of a novel envelope protein (VP281) of shrimp white spot syndrome virus by mass spectrometry

Abstract

The primary structure of a novel envelope protein from shrimp white spot syndrome virus (WSSV) was characterized using a combination of SDS–PAGE and mass spectrometry. The resulting amino acid sequence matched an open reading frame (ORF), ORF1050, of the WSSV genome ORF database. ORF1050 contained 843 nt, encoding 281 aa, and was termed the vp281 gene. Computer-assisted analysis showed that both the vp281 gene and its product shared no significant homology with other known viruses. However, they shared striking identity/similarity with another WSSV structural protein, VP292, at both the nucleotide and amino acid sequence level, suggesting that vp281 and vp292 might have evolved by gene duplication from a common ancestral gene. WSSV VP281 cDNA was cloned into a pET32a(+) expression vector containing a T7 RNA polymerase promoter to produce (His)6-tagged fusion proteins in Escherichia coli strain BL21. Specific mouse antibodies were raised using the purified fusion protein (His)6-VP281. Western blot analysis showed that the mouse anti-(His)6-VP281 antibodies bound specifically to VP281 of WSSV, without cross-reactivity with VP292. The transmission electron microscope immunogold-labelling method was used to localize VP281 in the WSSV virion as an envelope protein. The cell attachment ‘Arg–Gly–Asp’ motif in VP281 indicated that this protein might play an important role in mediating WSSV infectivity.