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机构地区:[1]中国水产科学研究院黄海水产研究所,青岛266071 [2]中国海洋大学,青岛266003
出 处:《水生生物学报》2006年第4期386-393,共8页Acta Hydrobiologica Sinica
基 金:国家重点基础研究规划项目(G1999012002)资助
摘 要:通过差速离心和蔗糖密度梯度离心,从感染了白斑综合症病毒(WSSV)的病虾头胸部分离了WSSV,利用地高辛对病毒蛋白进行了标记(DIG-WSSV),以体外培养的对虾血淋巴细胞为吸附基底,观察和分析了病毒与细胞间的结合现象及特性。以NBT/BCIP为酶反应显色底物观察到在细胞周围形成许多暗紫色颗粒,证实病毒与细胞间存在着稳定的结合。以OPD为酶反应显色底物分析了结合反应的特性:当DIG-WSSV维持恒定值时,随着血淋巴细胞数量的增加结合显色增强,细胞数量达到1.2×104cells/孔,492nm处的吸光值达到饱和;当血淋巴细胞数量维持恒定值时,随着DIG-WSSV蛋白含量的增加显色增强,且在DIG-WSSV的蛋白浓度达到4μg/孔时,492nm处的吸光值达到饱和;未标记WSSV可竞争抑制血淋巴细胞与DIG-WSSV间的结合作用。进一步的研究得出:4℃下,随着结合时间的延长显色增强,但继续延长结合时间显色反而减弱;缓冲液的渗透压对结合结果影响甚微,而酸性条件利于病毒与细胞间的结合。37℃孵育对病毒结合活性影响不大,55℃和70℃孵育可显著影响病毒的结合活性;短时间超声波处理病毒可增加病毒结合能力,长时间超声波处理可破坏病毒结合能力;有机溶剂处理同样可破坏病毒结合能力,其中尤以氯仿/甲醇的处理更为激烈;不同的去垢剂对病毒结合活性的影响结果不同:SDS和脱氧胆酸钠可以降低病毒的结合活性,而Triton X-100和NP-40可以提高病毒的结合活性。White spot syndrome virus (WSSV) was isolated from the cephalothorax of infected Penaeus chirvensis by differential centrifugation and then labeled by DIG-NHS in order to test whether it could bind to hemocytes of penaeid shrimp in vitro. Using an NBT/BCIP color detection system, dark purple grains were seen around cells by light microscopy and indicated stable binding between DIG-WSSV and hemocytes. Characterization of binding by holding the concentration of DIG-WSSV constant and measuring absorption at 492nm with a perroxidase (POD) detection system revealed that binding increased gradually as the quantity of hemocytes increased. Saturation was reached at 1.2×10^4 hemocytes per well. If the quantity of hemocytes was held constant, absorption (binding) increased gradually as the quantity of DIG-WSSV increased and saturation was reached at 4μg protein/well. Addition of sufficient unlabeled WSSV completely blocked this binding. Further analysis revealed that absorption at 4℃ increased with incubation time up to 40 min after which it decreased with prolonged incubation. Osmolarity of the binding buffer had little influence on binding while pH in the acid range promoted it. Incubation at 37℃ did not affect binding but at 55 and 70℃, it was reduced. Short term ultrasonic treatment increased binding but long term treatment reduced binding. Organic solvents reduced binding and this was particularly strong with chloroform/methyl alcohol. With respect to detergents, SDS and deoxycholic acid sodium salt decrease binding but Triton X-100 and Nonidet P-40 increased it.
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