【作者】 解飞霞；

【导师】 徐怀恕； 黄倢； 李筠；

【作者基本信息】 中国海洋大学 ， 海洋生物， 2003， 硕士

【摘要】 使用差速离心、蔗糖垫和蔗糖密度梯度离心法，从人工感染白斑综合症病毒(White spot syndrome virus，WSSV)的克氏原螯虾中提取WSSV，Bradford法测定病毒浓度为0.912mg／ml。分别取1ml的病毒悬液，用两种离子型表面活性剂SDS(十二烷基硫酸钠)和脱氧胆酸钠，及两种非离子型表面活性剂Triton X-100和Tween-20室温处理30min后，经不连续蔗糖梯度(40％—52％)离心和SDS-PAGE显示，确定这四种表面活性剂都可以增溶病毒囊膜的蛋白VP28。当病毒浓度为0.912mg／ml时，SDS、脱氧胆酸钠、Triton X-100和Tween-20增溶WSSV囊膜蛋白VP28的最佳浓度(v／v)分别为0.5％、1％、1％、1.5％。 地高辛(DIG,Digoxigenin-3-0-methylcarbonyl-e-aminocaproic acid-N-hydro xysuccinimide ester)标记完整的病毒粒子，标记产量为300pg／μl。标记的病毒用2％(v／v)非离子表面活性剂Tween-20增溶后，经阳离子交换层析(CM-sep harose)纯化，得到了电泳纯的病毒囊膜蛋白VP28。纯化的VP28与对虾鳃细胞膜的结合实验表明，VP28不能与对虾鳃细胞膜结合，即纯化的VP28不具有结合活性。 以健康对虾的鳃组织为材料，经过差速离心后获得对虾鳃细胞膜。测定蛋白浓度后，用地高辛进行标记，并测得标记后的产量为300pg／μL；SDS-PAGE得出对虾鳃细胞膜的多肽图谱；并与对虾白斑综合症病毒(White Spot Syndrome Virus，WSSV)进行体外结合实验，确定标记后的对虾鳃细胞膜具有结合活性。 对虾鳃细胞膜经过不同温度、pH、表面活性剂、无机盐离子浓度、DTT(二硫苏糖醇)、有机溶剂，及超声波处理后，再与WSSV结合。结果显示：对虾鳃细胞膜在25℃—45℃之间结合活性没有明显下降，但经煮沸5min后，结合活性丧失；pH的变化可以改变对虾鳃细胞膜的结合活性，其中在pH5和pH8时，活性比其它的pH值要高；NaCl不会使对虾鳃细胞膜的结合活性丧失，但NaCl浓度在1.5M和2.0M时可使其结合活性明显下降；硫酸铵在浓度为1M时对虾白斑综合症病毒仪SSV)囊膜蛋白vp28与对虾鳃细胞膜蛋白的分离和结合活性分析对虾鳃细胞膜的结合活性最高，在低于或高于IM时都会对虾鳃细胞膜的结合活性受到影响;超声波对虾鳃细胞膜的活性影响较大，作用时间505就可使对虾鳃细胞膜的活性丧失。共选用了5种表面活性剂处理对虾鳃细胞膜，包括Tween-Zo、NP并。、TritonX一00、脱氧胆酸钠、sDS(十二烷基磺酸钠)，作用浓度均为2%。前4种表面活性剂对结合活性有影响但不能使结合活性完全丧失，而SDS可使对虾鳃细胞膜的活性丧失。在上述各种表面活性剂中加入了0.IM二硫苏糖醇，实验结果区别很小，证实二硫苏糖醇对对虾鳃细胞膜的结合活性既没有起到保护的作用也没有起到破坏的作用。随着蔗糖浓度的加大，对虾鳃细胞膜的结合活性也随着下降，但是当蔗糖浓度达到62%时，对虾鳃细胞膜的结合活性仍然没有完全丧失。在有机溶剂中，三氯乙酸可使对虾鳃细胞膜的结合活性丧失，乙醇、丙酮可使对虾鳃细胞膜的结合活性有所下降，但不能完全丧失。 经2%Tween一20处理的对虾鳃细胞膜，CM一sephar0Se FF离子交换层析，得到3个洗脱峰;合并浓缩后测定与WSSV的结合活性，并将活性组分进行SDS一PAGE得到其蛋白多肤图谱。2%Tween一20处理后的对虾鳃细胞膜经过S叩hadexG一75凝胶过滤层析，浓缩后测定与wssv的结合活性，将得到的活性与蛋白量之比的最高峰进行SDS一队GE得到其蛋白多肚图谱。将离子交换的活性组分多肤图谱与凝胶过滤的活性组分多肤图谱相比较，得出分子量约为53KD的对虾鳃细胞膜蛋白具有与WSSV结合的活性。 将对虾鳃细胞膜与不同浓度的肝素钠混合，室温放置3h，发现对虾鳃细胞膜不再具有与WSSV结合的能力。将病毒与不同浓度的肝素钠混合放置同样的时间，却并未使病毒丧失与对虾鳃细胞膜结合的能力。从而推断肝素钠能够与病毒竞争对虾鳃细胞膜上的结合位点。更多还原

【Abstract】 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.更多还原