Cloning, Fusional Expression Of Envelope Protein Genes Vp19 And Vp28 Of Shrimp White Spot Syndrome Virus, And Preparation Of Neutralizing Antibody

In 1993, a disastrous infection in penaeid shrimp broke out in China, which induced by white spot syndrome virus (WSSV). It causes a high mortality of up to 90-100% in shrimps within 3-7days of infection. Outbreaks also occasionally took place in South America, Europe and Australia. It resulted in heavy economic losses for the world. Despite all the research efforts, up to date no effective measures or medicaments are available to prevent the spreading of WSSV. WSSV has a double-stranded DNA genome with a size of 305,107bp. The viral particle consists of at least five major proteins with estimated sizes of 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), 19 kDa (VP19), and 15 kDa (VP15). VP28 and VP19 are associated with the virion envelope and VP26, VP24, and VP15 with the nucleocapsid. The mechanism of virus entry into the shrimp and of the spread of the virus in the crustacean body is not known. And the physicochemical characterization and structure and functions of the viral genome and proteins of WSSV remain to be determined. Though shrimp cell line is not established for in vitro reproduction of larger amount of viruses, recently, a new WSSV-proliferating system using crayfish as a host instead of penaeid shrimp was established. The envelope of virus plays a key role in the process of viral infection and be somehow linked to the virulence of the virus itself. The role of the WSSV envelope and its proteins in the establishment of the systemic infection process has not been determined. Neutralization experiments have often been performed to study the role of virion proteins or their domains in the infection process. Neutralizing antibodies bind to envelope spikes on the virion and prevent attachment of the virus to the cell surface, cell entry, or virus uncoating. But little is known about the interactional mechanism between WSSV envelope and its cellular receptor now.At present, the in vivo neutralization experiments on WSSV in P. monodon with VP28 antibodies suggests that VP28 is located in the spikes of the WSSV envelope and this proteinmay thus be involved in the systemic infection of WSSV in shrimp. However, it can not be excluded that other WSSV envelope proteins, such as VP19, are also involved in this process, either alone or in concert with VP28.The Open Reading Frame(ORF) of vp19, vp28 envelope genes were amplified and linked by EcoR I restriction endonuclease site and then cloned into E.coli expression vector pET-22b(+) in the proper ORF. With IPTG induction at 35℃, the molecular weight of the engineering protein was about 41kDa which was identified by SDS-PAGE analysis. Antiserum of the fusion envelope protein VP (19+28), which was purified by Ni2+-column chromatography, was prepared by immunizing rabbit.The VP (19+28) antiserum was used to neutralize the WSSV infection in vivo of crayfish by intramuscular injection to identify its neutralizing efficiency. The results indicate that the antiserum was effective in the neutralization of WSSV on the crayfish which were reared with artificial food at 15-22℃. And it can also be concluded that the envelope proteins, VP28 and VP19, were involved in the process of WSSV infection. The high titer of the antiserum, on the other hand, is valuable for the development of diagnosis reagents and anti-WSSV medicines.