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Production and Preliminary Application of Monoclonal Antibodies Against Binding Protein of WSSV-VP37

Author: ChiZuoZuo
Tutor: ZhanWenBin
School: Ocean University of China
Course: Aquaculture
Keywords: White spot syndrome virus VP37-binding protein Monoclonal antibodies Litopenaeus vannamei Gill membrane proteins
CLC: S945
Type: Master's thesis
Year: 2011
Downloads: 18
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Abstract


White spot syndrome virus (WSSV) is the most important viral pathogen that causes considerable economic damage to shrimp culture industry. Since it outbroke in 1993, it has been studied by scholars at home and abroad. As the interaction of viral attachment proteins and the host cell receptors is the initiative and critical step of viral infection, therefore, studying the attachment proteins and the binding proteins of them on the host cell surface at molecular level that is very important to figure out the infection mechanism and search the strategies of prophylaxis and control of WSSV infection. VP37 is one of the main WSSV envelope protein, and it is reported that the antibodies against it have neutralization ability to WSSV infection, so VP37 might be an improtant adherency protein of WSSV. In this paper, the recombination VP37 protein was prokaryotic expressed, and the VP37-binding protein was identified in the gill membrane proteins (GMPs ) of L. vannamei by virus overlay protein binding assay (VOPBA). After electroelution, the VP37-binding protein was purified and identified as F1ATP synthaseβsubunit (β-F1ATPase) by Mass Spectromotry (MS). The purified VP37-binding protein was used to immunize Balb/C mice to produce monoclonal antibodies (Mabs), then the anti-VP37-binding protein Mabs were used to study the antigenic reactivity of gill tissues from different shrimps and the blocking activity of the binding of WSSV to the host cells in vitro. Specific contents and results are as follows:(1) Purified and identified of the VP37-binding protein. The VP37 gene with 843bp in length was amplified and inserted into pET32a plasmid, then transformed into E. coli BL21. The recombinant VP37(rVP37) were successfully expressed in positive clones with molecular masses of 52 kDa. Subsequently, the GMPs of L. vannamei were prepared by differential centrifugation and the VP37- binding protein was identified in the GMPs of L. vannamei by VOPBA. The result of VOPBA showed that rVP37 could interact with a 53 kDa protein band in GMPs. After electroelution, the 53 kDa protein band was purified and identified asβ-F1ATPase by MS.(2) Production of Mabs against the VP37-binding protein. The purified VP37-binding protein was used to immunize Balb/C mice to produce Mabs. After cell fusion, positive hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) and six of them designated as 1A5, 1D5, 1H5, 1E8, 2H4 and 2G5 were cloned by limiting dilution. Subsequently, the characteristic of the anti-VP37-binding protein Mabs was analyzed by indirect immunofluoresence assay (IIFA) and Western blot. The results of IIFA showed that Mabs could reacted with the gills of L. vannamei with specific fluorescence signals. Western-blot demonstrated that the Mabs reacted specifically with one protein band at the molecular mass of 53 kDa in GMPs of L. vannamei. Analyzed of their isotype, Mabs (1A5, 1H5, 1E8, 2H4 and 2G5) were of the subclass IgG and Mab 1D5 was of subclass IgM.(3) Preliminary application of Mabs against the VP37-binding protein. In the antigenic cross-reactivity assay, Mabs could cross-react with the gill tissues between L. vannamei and three other shrimps (Fenneropenaeus chinensis, Penaeus monodon and Marsupenaeus japonicus) by IIFA. It was inferred that three WSSV-susceptible shrimp gill cells might have the same antigenic epitope with VP37-binding protein on the GMPs of L. vannamei. Furthermore, anti- VP37-binding protein Mabs were tested in vitro for blocking activity against WSSV by ELISA, and the results showed that Mab 1E8、1H5 and 2H4 could partially block the binding of WSSV to GMPs of L. vannamei. These findings suggested that the VP37-binding proteinβ-F1ATPase involved in WSSV infection in shrimp L. vannamei.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Pest and Disease Control of the crustaceans
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