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引用本文:魏钰娟,潘晓艺,蔺凌云,姚嘉赟,郝贵杰,曹铮,夏焱春,尹文林,刘忆瀚,沈锦玉.异育银鲫(Carassius auratus gibelio)脊髓组织细胞系的建立及对CyHV-2的敏感性.海洋与湖沼,2020,51(5):1232-1238.
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异育银鲫(Carassius auratus gibelio)脊髓组织细胞系的建立及对CyHV-2的敏感性
魏钰娟1, 潘晓艺2, 蔺凌云2, 姚嘉赟2, 郝贵杰2, 曹铮2, 夏焱春2, 尹文林2, 刘忆瀚2, 沈锦玉1,2
1.上海海洋大学水产与生命学院 上海 201200;2.农业农村部淡水渔业健康养殖重点实验室 浙江省鱼类健康与营养重点实验室 浙江省淡水水产研究所 湖州 313001
摘要:
鲫造血器官坏死病是近些年来危害养殖鲫鱼最为严重的传染性疾病,其病原为鲤疱疹病毒II型(Cyprinid herpesvirus 2,CyHV-2)。本研究以异育银鲫(Carassius auratus gibelio)脊髓组织为材料,进行组织细胞体外培养,构建对鲤疱疹病毒II型敏感的异育银鲫脊髓细胞系(Spinal cord tissue cell lines of Carassius auratus gibelio,CSC)。采用组织块移植法,进行异育银鲫脊髓细胞的原代培养,并进行传代,已传代至第128代次,获得稳定传代的细胞系后,采用低渗法进行细胞染色体核型分析,并进行鲤疱疹病毒II型的敏感性测试与传代。结果表明,以含10%胎牛血清的L-15培养基,在24℃恒温条件下,可稳定传代培养异育银鲫脊髓细胞,传代细胞呈成纤维样或纺锤状;核型分析显示,染色体数为156±2条,该细胞为三倍体细胞。通过对CyHV-2敏感性的测试,CSC细胞感染CyHV-2后产生典型的细胞病变,感染第7天的病变细胞液的TCID50达到109.33/mL,CyHV-2病毒可在CSC细胞上稳定传代;通过对病变细胞的电镜观察,可见大量直径为128—134nm大小的疱疹样病毒颗粒。以上研究结果表明,本研究构建的异育银鲫脊髓细胞系对CyHV-2敏感,并在细胞上能进行稳定传代,传代病毒具有较高病毒滴度,这为鲤疱疹病毒II型的深入研究及疫苗开发奠定了重要的基础。
关键词:  异育银鲫(Carassius auratus gibelio)  脊髓组织细胞系  鲤疱疹病毒II型
DOI:10.11693/hyhz20200300071
分类号:S943
基金项目:浙江省重点研发计划,2019C02058号;浙江省省属科研院所扶持专项,2020YSZX001号
ESTABLISHMENT OF SPINAL CORD CELL LINE OF CARASSIUS AURATUS GIBELIO AND ITS SENSITIVITY TO CyHV-2
WEI Yu-Juan1, PAN Xiao-Yi2, LIN Ling-Yun2, YAO Jia-Yun2, HAO Gui-Jie2, CAO Zheng2, XIA Yan-Chun2, YIN Wen-Lin2, LIU Yi-Han2, SEHN Jin-Yu1,2
1.School of Fisheries and Life, Shanghai Ocean University, Shanghai 201200, China;2.Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs, Key Laboratory of Fish Health and Nutrition of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China
Abstract:
Hemopoietic organ necrosis of Crucian carp (Carassius auratus gibelio) is the most serious disease causing economic loss of Crucian carp. In this study, the spinal cord tissue cell line of C. auratus gibelio, named CSC was established in vitro. The spinal cord cells of crucian carp were cultured in primary culture by tissue block transplantation, and passed on. After obtaining stable cell lines, the cell chromosome karyotype was analyzed by hypotonic method, and the sensitivity test and passage of carp herpesvirus type II were carried out. The results show that the CSC could be stably subcultured in L-15 medium containing 10% fetal bovine serum at 24℃, and the subculture cells are mainly composed of fibroblast-like and spindle-like cells. Karyotype analysis showed that the number of chromosomes was 156±2, and the cells were triploid. Through the test of CyHV-2 sensitivity, CSC cells infected with CyHV-2 produced typical cytopathic effects. On the 7th day of infection, TCID50 of the cells infected with CyHV-2 reached 109.33/mL, and CyHV-2 virus could be continuously and stably passaged. A large number of herpes-like virus particles with a diameter of 128—134nm in the cells infected with CyHV-2 were observed with electron microscope. Results show that the spinal cord cell line of C. auratus gibelio constructed in this study was sensitive to CyHV-2 and could be stably subcultured. The subculture virus showed a high virus titer, which lays an important foundation for the further research and vaccine development against Cyprinid herpesvirus 2.
Key words:  Carassius auratus gibelio  spinal cord tissue cell line  CyHV-2
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