Abstract
White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV.
Highlights
White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) utilizing a quadrupole time-of-flight (Q-TOF) mass spectrometer have been used as tools for the characterization of proteins because of their high sensitivity and throughput [10]
WSSV is a major pathogen with a broad range of hosts, high infectivity, and high mortality
Summary
Proliferation and Purification of WSSV Virion—The infected tissues from penaeid shrimp P. monodon (e.g. gill, stomach, midgut, etc.). Proteomic Analysis of Shrimp White Spot Syndrome Virus were homogenized in TN buffer (20 mM Tris-HCl and 400 mM NaCl, pH 7.4) at 0.1 g/ml. Virus bands were collected by side puncture, diluted 1:10 with TNE buffer (50 mM Tris-HCl, 100 mM NaCl, and 1 mM EDTA, pH 7.4), and pelleted at 119,000 ϫ g for 1 h at 4 °C. Purification of WSSV Nucleocapsid—Purified WSSV virion was treated with Triton X-100 for 15 min at room temperature, subjected to 20 –50% continuous CsCl gradient, and centrifuged for 24 – 48 h at 110,000 ϫ g using a SW 41-Ti. Viral capsid band was collected by side puncture and diluted with 1ϫ TN buffer (1:10), subsequently sedimented at 120,000 ϫ g for 45 min. The pellet was resuspended in 1ϫ TN buffer, pH 7.4
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