| 摘要: |
| 染病中国对虾于1995年7月取自青岛崂山对虾养殖场。为探索对虾杆状病毒分离纯化技术，采用组织超薄切片技术对染病对虾进行电镜观察；被病毒感染的对虾经组织匀浆、差速离心和密度梯度离心。纯化出杆状病毒粒子；纯化的杆状病毒经磷钨酸钾负染色，于透射电子显微镜下观察形态结构。结果表明，中国对虾中肠组织的细胞核被大量的有囊膜无包涵体的杆状病毒侵染，属C型杆状病毒；病虾头部组织以6000 r/min匀浆，6000 r/min与10000 r/min差速离心，再经10％－50％蔗糖密度梯度，可纯化出形态完整的杆状病毒粒子；与所有已报道的对虾杆状病毒不同，中国对虾完整的病毒粒子带有一很长的尾，可达病毒体的1.5倍以上。经Tritonx-100处理，获得脱去囊膜的核衣壳，衣壳系由两个帽端夹14圈螺旋形成对称的圆柱体结构。 |
| 关键词: 中国对虾 病毒 纯化 |
| DOI： |
| 分类号: |
| 基金项目:国家攀登计划“B”资助项目，PDB6－6－1号 |
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| STUDIES ON PURIFICATION TECHNIQUE AND MORPHOLOGY OF A KIND OF TYPE C BACULOVIRUS IN PENAEUS CHINENSIS |
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Kong Jie1, Shi Tuo1, Liu Ping1, Bao Zhenmin2, Guo Fusheng3, Xiang Jianhai4
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1.Yellow Sea Fisheries Research Institute, Chinese Acadimy of Fishery Sciences, Qingdao 266071;2.College of Life Sciences,Ocean University of Qingdao,266003;3.National Animal Quarantine Institute, Ministry of Agriculture, Qingdao 266071;4.Institute of Oceanology , Chinese Academy of Sciences, Qingdao 266071
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| Abstract: |
| Moribund shrimps, Penaeus chinensis (3 - 5 cm in body length), were collected during an occurrence of mass mortality at a shrimp farm of Laoshan, Qingdao in July 1995- The gill tissue, hepatopancreatic tissue and mid-gut tissue were fixated for ultrathin sectioning and the existence of a kind of baculovirus was found in the gill and mid-gut by Hitachi H-7000 transmission electron microscope. Cytopathic changes associated with baculovirus infection were hypertrophy of the nucleus and accumulation of rod-shaped enveloped virions (Plate I:1). To isolate the virus, 100g of moribund shrimp were homogenized at 6000 r/min for 5min with PPB buffer (Huang Jie, personal communication) or PPB buffer containing 2% Triton X-100. The outer membrane of the virions was broken and the virions were purified as nucleocapsids in the presence of Trition X-100. Pellets of the material homogenated at different centrifugation speeds were inspected by electron microscope and baculovirus was found to be precipitated at 8000 r/min (Tab. 1). Morphologically intact baculovirus and nucleocapsids could be isolated and purified by centrifugation from 6000 r/min to 10000 r/min and density gradient centrifugation on 10%-50% sugrose at 25000 r/min from cephalothorax homogenate. The morphology and structure of the virus, stained by phosphotungstic acid (PTA), were analyzed by transmission electron microscope. Morphologically intact baculovirus were rod-shaped, singly enveloped and with a long tail-like appendage. The head of whole virions averaged 90-130 nm in diameter and 300-370 nm in length. The Tail was 20 - 30 nm in diameter and 500 - 700 nm in length (Plate I: 2). The nucleocapsid contained 14 helixes with 2 capped ends on both sides and formed bacilliform structure (Plate I: 3). |
| Key words: Peneaus chinensis, Virus, Purification |
