| In this thesis, ascites were produced and purified utilizing hybridomas which secrete monoclonal antibodies (MAbs) against the envelope proteins VP28, VP26 or VP19 of White Spot Syndrome Virus (WSSV). The neutralization effects of mixed monoclonal antibodies and MAbs against WSSV of different concentrations were analyzed utilizing ELISA and crayfish (Cambarus proclarkii). These results demonstrated that the neutralization effect of MAb1D6 was WSSV-concentration dependent. Additionally, VP28, VP26 and VP19 were isolated through SDS-PAGE and then analyzed employing 2D gel electrophoresis. The followings are the details.(1) Purification of WSSV and production of specific MAbs against WSSV envelope proteins: WSSV was isolated by differential centrifugation and density gradient centrifugation. Four MAbs specifically against WSSV envelope protein, respectively named 1D6, 2G3, 3G7 and 2E9, were purified from ascites that were produced through inject resuscitated hybridomas into Balb/c mouse. Resluts of indirect immunofluoresence assay (IFA) proved that all of the four MAbs reacted with native WSSV in gill slices of infected shrimp. Through western-blot assay, both 1D6 and 2G3 were demonstrated to recognize VP28, while 3G7 was identified to react with VP26 and 2E9 was identified to react with VP19.(2) In vivo neutralization assay of MAbs against WSSV of different concentrations: The optical density (OD) values of ELISA which reflect the combination between WSSV and MAbs showed that both MAb1D6 and MAb2E9 were sufficient at 1×10-3 virus dilution. In vivo neutralization assays were carried out in the animal model of crayfishe (C. proclarkii). For each crayfish, 50μL WSSV at different dilutions were pre-incubated with MAb and intramuscular injected, after which the cumulative mortalities of each groups were recorded. Results showed that mortalities in the MAb1D6 group were 100%, 90%, 16.7% and 6.7% at dilutions of 1×10-3, 1×10-4, 1×10-5 and 1×10-6 respectively, while the corresponding mortalities in the MAb2E9 group were respectively 100%, 100%, 100% and 93.3%. These results demonstrated that the neutralization effect of MAb1D6 was concentration dependent while MAb2E9 had no obvious neutralization effect. IFA confirmed that the crayfishes died in the in vivo neutralization assay were all WSSV infection positive except ones from the MAb1D6 neutralized groups with WSSV dilutions of 10-5 and10-6 and the negative control group.(3) The neutralization of WSSV by mixed MAbs: Digoxingenin (DIG) labeled WSSVs (DIG-WSSV) were incubated with MAbs before added to ELISA plates that were coated with haemocyte membranes isolated from Fenneropenaeus chinensis.The combination of DIG-WSSVs to haemocyte membranes were then detected by anti-DIG antibody. There was no different in the optical density (OD) values between groups incubated with single MAb and groups incubated with mixed MAbs, which implied that no cooperated neutralization effect existed between MAbs. In the crayfish in vivo neutralization assay, no difference of cumulative mortality was observed between group neutralizes only by MAb1D6 and groups respectively neutralizes by mixture of MAb1D6 and one of the other three Mabs, which confirmed the absence of cooperated neutralization effect between MAb1D6 and other MAbs. (4) Analysis of WSSV envelope protein VP28: WSSV proteins VP28, VP26 and VP19 were isolated through SDS-PAGE and electro-elution. Western-blot analyses demonstrated that the isolatated VP28 reacted with both MAb1D6 and MAb2G3. After 2D gel electrophoresis, only one protein dot with a pI of 5.8 were identified from VP28 by blue silver staining, which indicated that VP28 was made up of one single kind of protein.
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