Studies On Invasion Mechanism Of Shrimp White Spot Virus And Its Specific Single-Chain Antibody Genes

White spot syndrome virus (WSSV), a penaeid shrimp virus, caused significant economic loss in the shrimp industry around the world. A major outbreak of white spot disease occurred in China in 1993, and resulted in a 70% reduction in shrimp production in a single year. Until now, the molecular mechanism of viral infection is poorly understood and, no effective drug has been developed. It still makes control of white spot disease difficult. Meanwhile, there is no continuous and stable cell line for WSSV inoculation and propagation, it makes WSSV study very difficult, especially on the function of genes. This thesis was just focused on these aspects based on the purified WSSV virions: 1) to study the cell culturing for WSSV inoculation and propagation; 2) to identify the function of VP28 protein by using the E.coli-expressed VP28 and VP28-EGFP (tagged with EGFP protein) in the cellular experiments. 3) by using phage display technique, the phage-displayed antibodies which can specifically bind to WSSV were selected, it can be used in the WSSV detection and neutralized antibody development in the future study.Primary shrimp cell culture from lymphoid organs (Oka) of Peneaus monodon was successfully developmented with 15% fetal bovine serum, L-15 culture medium and a salt mixture. Plates were then incubated at 28℃ until 70-80% cell monolayers were formed. Even though these primary lymphoid cell cultures could not be subcultured, they proved to be useful for studying shrimp virus which could induce unequivocal cytopathology in them. Cytopathic effect (CPE) induced by white spot virus showed characteristics of cell rounding, detachment and lysis. Quantitative analysis of WSSV infected P. Monodon was determined using primary lymphoid cell systems and the titer was 3.23 ×10⁷ TCID₅₀/ml by using TCID₅₀ method.The cells liabilities at different of concentrations of WSSV infected cell cultures was determined, and the results showed that there was a negative relativity between WSSV concentrations and liabilities. Also, the baculo-virus virions were observed by binding negative-dyeing and electromicroscopy in the cells culture, in which 10" WSSV cells culture infected again. Meanwhile, in the ultrathin section of cells infected with WSSV, the virions were also detected in the cytoplasm. Thus all of the above evidences showed that the primary lymphoid cells culture was fitted for WSSV propagation and also very useful for studying shrimp virus other than WSSV.A chimeric protein VP28-EGFP was successfully expressed with a green fluorescence marker by fusing vp28 gene with egfp gene. The significant green fluorescence was detected by microscopy. The chimeric protein was mixed into primary cell culture at different of concentrations. In this investigation, the observation of binding of VP28-EGFP to cells gave us the direct evidence that VP28-EGFP could bind with shrimp cells at pH 6.0 within 1 h in the binding assay in vitro. The membrane protein was extracted for further Western blotting analysis, and the result showed that VP28-EGFP was specially bound with cell membrane. By using the laser scanning confocal microscopy, the VP28-EGFP binding cells showed the different fluorescence with the different time after WSSV infected. Within 1 h the protein showed a "fluorescence ring" around cell membrane, but the fluorescence enter into cytoplasm at 3 h post-adsorption.The antibody blocking test was carried by using the polyclonal antibody against VP28. The result showed that the polyclonal antibody against VP28 could neutralize WSSV and block the viral infection. On the other hand, the result of competition ELISA further confirmed that the envelope protein VP28 could compete with WSSV to bind to shrimp cells. The above evidences indicated that there is a probable acceptor existing on the cell membrane, and which could be blocked by antibody against VP28 also could be competed with VP28 in the process of WSSV infecting shrimp cells and binding to cells acceptor. Thus, it was concluded that the envelope protein VP28 of WSSV could bind to shrimp cells as an attachment protein and could help virus entry into cytoplasm.In addition, the phage display antibody library was screened against purified WSSV and, five specific antibodies (scFv) were selected. The sequence analysis showed that all of the five antibody genes were composed of VH gene (345bp) and Vk gene (384bp). There are high homologies of VH genes that reached 90% among the five antibody genes, but the similarities of VK genes of the five arrived at 98%. Of the fiveantibody genes, two from library I are very similar. However, the other three from library J keep the high variability. The character of these antibodies, the inhibition efficiency to WSSV is being further studied.