| WSSV(white spot syndrome virus) is isolated from infected crayfish cambarus clarki., by different speed centrifugation , sucrose cushion , sucrose gradient centrifugation. Quantified by Bradford methods, the concentration of WSSV is 0.912mg/ml. WSSV is treated 30min with two ionic detergents, SDS and Deoxycholic Acid Sodium, and two non-ionic detergents, Triton X-100 and Tween-20. Following with discontinuous sucrose gradient (42%-50%) centrifugation, the result was shown on SDS-PAGE. Those detergents can solubilize vp28, which is a characteristic envelope protein of WSSV. When the concentration of WSSV is 0.912mg/ml, the concentration(v/v) of SDS, Deoxycholic Acid Sodium, Triton X-100 and Tween-20 is 0.5%, 1%, 1%, 1 .5% separately.WSSV is labeled by DIG (Digoxigenin-3-O-methylcarbonyl-e-aminocaproicacid-N-hydroxysuccinimide ester), and the labeling quality is 300 pg/ul. After dissolving DIG- WSSV by 2% Tween-20, Vp28 can be purified by CM ion-exchange chromatography. Binding assay shown that Vp28 can not binding membrane protein of shrimp gill.Shrimp cell membrane was obtained from healthy shrimp gill through centrifugation. After quantifying its concentration by Bradford methods, it is labeled by DIG and the labeling quality is 300 pg/ l. It is confirmed that the cell membrane of shrimp gill can bind to virus in vitro. The polypeptide map of cell membrane of shrimp was made by SDS-PAGE.The cell membrane of shrimp was treated by different conditions, such as temperature, pH, detergents, ion concentration , DTT, solvent and ultrasonic. Then the cell membrane was bind with WSSV in vitro and the binding assay result shown that: 1) Along with temperature rising from 25 to 45 , the binding activity of cell membrane do not decline distinctly. After the membrane was boiled 5min, its binding activity would lose completely. 2) The variation of pH can not make the binding activity of cell membrane lost, and it got highest value when the pH is 5 and 8. 3) When the concentration of NaCl is 1.5M or 2.0M, the binding activity of cell membrane of shrimp decline distinctly. But NaCl cannot make its binding activity lost. The concentration of (NH4)2SO4 would influence the binding activity of cell membrane of shrimp, and it is the highest when the concentrations of (NH4)2SO4 is 1M. 4) The binding activity of cell membrane of shrimp may be influenced distinctly, if it was treated by ultrasonic. Such as if the treated time only is 50s, the binding activity would lose completely. 5) The cell membrane was treated by different detergents, and the concentration of detergents is 2%. Tween-20, Np-40, Triton X-100 could not influence the binding activity, and SDS make binding activity lost completely. 6) DTT does not have any influence to binding activity. 7) Along with the rising of sucrose concentration ,the binding activity is declining but can not lose. 8) TCA make binding activity losed completely, alcohol and acetone declined the binding activity of cell membrane.Treated with 2% Tween-20 and purified by CM-Sepharose FF ion exchange Chromatography, cell membrane of shrimp was separated to three fractions. After concentrating and binding to virus, the third fraction has the binding activity. The polypeptide map was obtained through SDS-PAGE. Treated with 2% Tween-20 and purified by Sephadex G-75 gel filtration, cell membrane of shrimp was separated to several fractions. After concentrating and binding to virus, gained the fraction that the ratio of binding activity and quality of protein is the highest. The polypeptide map of the fraction was obtained through SDS-PAGE. Compared two polypeptide maps, it is found that the 53kD protein has binding activity.Blended with different concentration of sodium heparin (under room temperature 3 hr), shrimp cell membrane loses binding activity and the best concentration is 40mg/ml. That is sodium heparin can block the virus-binding site of cell membrane of shrimp.
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