牙鲆淋巴囊肿病毒纯化及不同来源病毒免疫特性比较

Purification and western blotting of three strains of lymphocystis disease virus

关键词:  淋巴囊肿病毒  超速离心  分离纯化  免疫特性比较

Key words:  Lymphocystis disease virus (LCDV)  Ultracentrifugation  Purification  Immunological characterization

摘要:

比较了3种不同的牙鲆淋巴囊肿病毒纯化方法，优化后的方法如下：剥离囊肿表面薄膜，收集内容物，匀浆后再用超声波细胞破碎仪破碎，反复冻融，650×g、1 800×g差速离心，30%（W/W）蔗糖垫底超速离心（78 500×g）浓缩病毒，最后蔗糖密度梯度超速离心（78 500×g）纯化病毒。电镜观察发现，出现在47%-52%蔗糖密度区域的病毒带含有多量、纯净和结构一致的病毒粒子。此外，利用制备的兔抗血清对不同地区的病毒进行了免疫特性分析，Western blotting检测显示来自威海、青岛及秦皇岛3个地区的淋巴囊肿病毒反应结果是一致的，均有3条蛋白带发生反应，其分子量分别为125、66和55kDa。

Abstract:

The diseased Japanese flounders Paralichthys olivaceus were obtained from a culture farm near Qingdao. The lymphocystis nodules were cut down and washed with TNE buffer (50mmol/L Tris, 100mmol/L, 1mmol/L EDTA, pH 7.4), about 10 gof nodules were homogenized and suspended in 90 ml TNE buffer at 4℃. The suspension was subjected to three rapid freeze/thaw cycles. Following ultrasonication, the virus cell debris suspension was centrifuged at 650×g for 20 min at 4 ℃. Then the supernatant was centrifuged again at 1 800×g for 20 min at 4 ℃, pooled and stored overnight at 4 ℃, while the pellet was discarded. Virus particles were pelleted from the pooled supernatant by ultracentrifugation at 78 500×g for 120 min at 4 ℃ and resuspended in TNE. The suspension was equally divided and loaded onto pre formed gradients made up of 33%, 40%, 47%, 52%, 57% and 62% (w/w) sucrose, and ultracentrifuged at 78 500×g for 120 min at 4 ℃. Two prominent bands were obtained in sucrose gradients after ultracentrifugation. A diffuse band (B1) was located at about 37%~40% sucrose concentration, while a distinct band (B2) was located at about 47%-50% sucrose concentration. B1 contained few viral particles together with amorphous material, membranous structure and other cell debris when assessed by electron microscopy, while B2 from sucrose gradients contained viral particles and was apparently free of other material. In electron micrographs, the virions were intact, round polygon in shape, about 210-250 nm in diameter, some of the viral particles appeared with a more or less hexagonal profile.  In addition, for learning immunological characterization of LCDV purifitied from different zones，the rabbit antiserum was raised against the LCDV purified from Weihai region. Serum homology of LCDV isolates from Qin Huangdao, Qingdao and Weihai were checked by western blotting. Western blotting profile showed three main antigenic proteins with molecular weights of 125, 66 and 55kDa reacted with the antiserum. This experiment showed that the virus from different zones had the same linear epitopes.