Functionally Identification Of Non-specific Nuclease And Construction Protein VP31 From White Spot Syndrome Virus (WSSV)

White spot syndrome virus (WSSV) is one of major pathogens in the cultured penaeid shrimp.This paper is concerned on the possible pathogen related gene, two main works are included.(1) Functionally identification of non-specific nuclease from WSSV. In this section, The product encoded by the wsv191 gene from shrimp white spot syndrome virus (WSSV) is homologous with non-specific nucleases (NSN) of other organisms. To functionally identify the protein, the wsv191 gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with 6His-tag at C-terminal. The fusion protein (termed as rWSSV-NSN) was purified using Ni-NTA affinity chromatography under denatured conditions, renatured and characterized by three methods. The results showed that rWSSV-NSN could hydrolyze both DNA and RNA. 5'-RACE result revealed that the transcription initiation site of the wsv191 gene was located at nucleotide residue G of the predicted ATG triplet. Therefore we concluded that the next ATG should be the genuine translation initiation codon of the wsv191gene. Western blot analysis revealed that the molecular mass of natural WSSV-NSN was 37 kDa.(2) Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31.In this section, based on a combination of SDS-PAGE and mass spectrometry, a protein with an apparent molecular mass of 31 KDa (termed as VP31) was identified from purified shrimp white spot syndrome virus (WSSV) envelope fraction. The resulting amino acid (aa) sequence matched an open reading frame (WSV340) of the WSSV genome. This ORF contained 783 nucleotides (nt), encoding 261 aa. A fragment of WSV340 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with a 6His-tag and then specific antibody was raised. Western blot analysis and the immuno-electron microscope method (IEM) confirmed that VP31 was present exclusively in the viral envelope fraction. By using a fluorescent probe method, we have investigated the interaction between protein and host cells. RGD competitionexperiments showed the presence of known competitive peptide (RGD) could block cellular adhesion with protein relative to the control (noncompetitive peptide RDG). Therefore, that RGD play an important role in interaction host cell with VP31.The neutralization experiment suggested that VP31 might play an important role in WSSV infectivity.