Function Analysis Of One Structural Protein Gene From White Spot Syndrome Virus And Construction Of The Recombinant Virus

White Spot Syndrome Virus (WSSV) is the main viral pathogen of Penaeid shrimp. It seriously affects the shrimp production and quality worldwide since it was first found in the early of 1990s. First of all, this thesis introduced the important viral pathogens reported in shrimp aquaculture, especially, in detail on the progress in WSSV studies (see chapter 1).WSSV possess the envelope protein that was easily destroyed during the virus purification. We thus developed a modified protocol based on routine sucrose gradient centrifugation. By adding the citrate sodium to the sucrose gradient, we can obtain a large quantity of purified intact virions. The SDS-PAGE and Western blot results showed that this method is very useful and efficient for WSSV purification (see chapter 2).The particularity of WSSV genome and the shortage of WSSV permissive cell line retarded the gene function studies. Only few gene's function have been investigated. In chapter 3 and 4, one ORF of WSSV (ORF220) was analyzed and expressed. The specific antibody was subsequently produced against the expressed protein. Sequence analysis showed that this putative gene contains the conserved motif of eukaryotic cytokine receptor gp130 and has a theoretical molecular weight of 76 kD (designed as VP76). When analysed by SDS-PAGE, both of intact virions and envelope fraction present a weak band of 76 kD. The western blot with anti-VP76 antibody also demonstrated that the VP76 was an envelope protein. To investigate the function of this protein, WSSV was neutralized with the VP76 specific antiserum in different concentration and then injected intramuscularly into crayfishes. The mortality curves showed that the VP76 antiserum could attenuate the infection of WSSV. This suggested that VP76 was an envelope protein involved in WSSV infection.Based on homologous recombination, the replicon of E. coli was inserted into virus genome and thus made the virus replicate in both of prokaryote and eukaryote cell. This kind of recombinant virus was very useful for virus gene function studies. Here we have made the preliminary progress on constructing the WSSV recombinant virus. We succeeded in demonstrating the infectivity of WSSV genome and constructing two transfer vectors, whichprovide a good background for further recombinant virus construction.