Against Recombinant Vp28 Capsule Membrane Protein Of Monoclonal Antibodies In The Detection Of White Spot Syndrome Virus

White spot syndrome (WSS) is an acute and highly contagious disease caused by white spot syndrome virus (WSSV), which causes great economic losses to the shrimp industry. Due to the lack of adaptive immune mechanism in crustaceans, all the efforts are directed towards the development of diagnostic tests for efficient detection of WSSV. The trial was conducted to express the WSSV envelope protein VP28 gene in prokaryote system and preparation the monoclonal antibodies (MAbs). The result of this study will provide technical support to detect the shrimp infection of WSSV and it will be significant in the prevention and control of the virus. The main research contents and results were as follows:1) The virus we isolated from infected crayfish was coated with capsule and was about 100～120 nm in diameter and 300～400 run in length. The uncleocapsid without the capsule was about 80～90 nm in diameter and 300～450 nm in length, which was spiral-shaped cylinder. The morphate and size of virus we purified was similar to WSSV as reported before.2) Envelope protein VP28 gene of WSSV was amplified by PCR. Cloned VP28 gene was expressed in E.coli and purified by Ni-NTA column. The induced products were analyzed by SDS-PAGE and Western Blot. The resluts showed that the band corresponding to VP28 was observed at the expected height, indicating that VP28 was expressed in E.coli successfully.3) Purified recombinant VP28 was used to immunize Balb/c mice. The immunized mice's spleen cells and their myeloma cells ware then conjugated. After that we screened out the positive hybridoma cells, and cloned a hybridoma cell strain, which could steadily secrete sharply positive MAbs. Immuno-electron microscopy and ELISA was employed to detect the specificity. The gold particles were distributed in the outer envelope of WSSV virions. The results not only proved that VP28 is a virus envelope protein, but also showed that anti-rVP28 MAb could recognise native VP28 target epitopes. It implied that it was possible to detect WSSV with anti-rVP28 MAb. 4) Anti-rVP28 MAb can be used to detect natural WSSV infection by dot-blotting analysis. Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay. It suggested dot-blot analysis with anti-rVP28 MAb can detect WSSV rapidly and sensitively at the early stages of WSSV infection.