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White spot syndrome virus (WSSV) is a major pathogen in the cultivated shrimp. Nine homologous repeat regions (hrrs) have been reported interspersed throughout the WSSV genome. In this investigation, the protein fraction that specifically bound to the hrrs was isolated by using DNA-affinity chromatography. A total of 9 (S1 to S9) and 5 (C1 to C5) proteins separated from the WSSV infected shrimp and the healthy shrimp, respectively, were detected by using two-dimensional polyacrylamide gel electrophoresis, and 6 proteins changed with WSSV infection were analyzed by mass spectrometry (MS). One (S4) of the 6 proteins examined was identified as WSSV ORF59 protein, and another (S3) was a shrimp arginine kinase. No homologous proteins were found with the remaining 4 proteins by searching in the WSSV ORF database and NCBI database. The specific binding site of the 6 proteins was then determined by gel mobility shift assay (GMSA). Temporal analysis revealed that ORF59 gene was transcribed at the early stage of the infection. The results we obtained provide important information to understand WSSV replication. The combination of DNA-affinity chromatography, 2D-PAGE and MS approaches should have general application to the identification of gene regulating proteins of WSSV. The results represent the first isolation of a set of proteins that bind to the hrrs, and, furthermore, lead us a new research direction for the prevention and the therapy of WSSV.

Proteomic Analysis of Proteins that Binds Specifically to the Homologous Repeat Regions of White Spot Syndrome Virus