| Since its first appearance in1987, White spot disease (WSD) had caused serious economic losses all over the world. After that, the study on its causative agent——White spot syndrome virus (WSSV) never ended. The WSSV is a large, nonoccluded, enveloped, rod-or elliptical-shaped, doubled-stranded DNA virus of approximately300kbp. It belongs to the Nimaviridae family, and is a major pathogen in the cultured penaeid shrimp. It has a wide range of hosts in crustaceans, a high infection and mortality rate, and is hard to be controlled. Therefore, WSSV is not only a major threat to the shrimp industry but also to the water ecology and environment. Like the other viruses, the study on the structural proteins of WSSV is always a hot field. This thesis mainly focuses on the function of virus structural proteins. In addition, the author also tried to study the function of the IE genes using RNAi analysis in this thesis. The work mainly includes the following aspects:(1) VP12protein, the product of the wsv009gene of white spot syndrome virus (WSSV), is one of the low-abundance structural proteins of virus. In our previous studies, VP12was found to exist in the envelope part. To confirm it, VP12was cloned and expressed in Escherichia coli and its polyclonal antiserum was prepaired using the purified recombinant VP12. Then using its antiserum, western blotting analysis and immunoelectron microscopy were performed. The result indicated that VP12behaved as an envelope protein and it was assumed to lie inside the envelope. In the following studies, we confirmed the VP12/VP150complex does exist firstly. Secondly, the interaction between VP12and VP51was studied under non-natural conditions and then, Far-Western blotting analyses provided further evidence for direct interaction between VP12and VP51. On one hand, these results further suggest that VP12lie inside the envelope. On the other hand, these findings suggest that VP12, like VP26, may also function as a linker protein participating in the linkage between VP12/VP150complex and viral nucleocapsid. Later, VP12was found to interact with VP26and the self-polymerization of VP12through its intermolecular disulfide bond was confirmed too. It means VP12could form a interactional meshwork that lies between the envelope and nucleocapsids together with the proved linker protein VP26. And the formation and existence of this meshwork may be important to keep the stability of the virions.(2) Due to the three-dimensional network of the protein interaction, we attempted to find which envelope protein(s) that the VP12/VP150complex bound to after the work has been done in Part1. Through CO-IP assay, VP28, one of the major envelope protein, was found to interact with the N terminal (aa1-206) of VP150. The result of Far-western blotting assay confirmed that VP28does bind to the undegraded form of VP150. This finding suggests that the degradation of VP150may starts from its N terminal. More studies will be needed to prove this speculation in the future. Combined with the results gained in Part1, it could be seen that though the ineraction between VP150and VP28, VP12/VP150complex could bound to other larger multi-protein complex. Thus, a three-dimensional network stretched across envelope and nucleocapsid was formed.(3) Since IE genes encode proteins involved in activating the expression of viral early and late genes, altering the functions of host genes and eliminating host immune defense, their expression is especially important in determining host range. To reveal the function of IE, RNA interference (RNAi, a process by which dsRNA directs sequence-specific degradation of messenger RNA) was employed. In this study, freshwater crayfish, Procambarus clarkii, were intramuscularly injected with double-stranded RNA(dsRNA) corresponding to7IE genes of WSSV (wsv051、 wsv056、wsv06、wsv079、wsv083、wsv100、wsv249) and an unrelated dsRNA of a green fluorescence protein (GFP) gene. Quantitative RT-PCR analysis showed that gene-specific dsRNA could result in higher inhibition of WSSV replication, compared with the GFP group and positive control (P<0.05). RT-PCR was carried out to further determine the suppressive degree of dsRNA corresponding to the7IE genes. The results showed that wsv051-dsRNA and wsv083-dsRNA had relatively more efficiency in inhibiting WSSV transcription among the7IE genes selected. These results suggest that RNAi should be feasible in studing the function of the IE genes. Our attempts provided important experience and idea for the future research.In conclusion, it is meaningful to study the properties and interactions between envelope proteins which may help us to better understand the invasion, assembly, maturation and release mechanisms of WSSV. Further more, exploration of exact molecular mechanisms of virion morphogenesis will lay the scientific basis for WSSV prevention. |
PDF Full Text Request