| 大口黑鲈弹状病毒的分离培养及其卵黄抗体的制备 |
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| 作者姓名： | 袁雪梅 吕孙建 施伟达 杭小英 刘莉 吴颖蕾 |
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| 作者单位： | 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 |
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| 基金项目： | 湖州市自然科学资金项目(2018YZ09);浙江省省属科研院所扶持专项项目(2019YSZX002)共同资助。 |
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| 摘 要： | 本研究从爆发性死亡的大口黑鲈(Micropterus salmoides)鱼苗病样中分离培养1株大口黑鲈弹状病毒(M.salmoides rhabdovirus,MSRV)毒株,采用甲醛灭活大口黑鲈弹状病毒,制备佐剂灭活疫苗,免疫产蛋鸡,收集高免蛋,制备MSRV卵黄抗体,测定其效价、中和效果及对病毒复制和宿主细胞内凝集素基因(intelectin)表达的影响。结果显示,成功利用草鱼(Ctenopharyngodon idellus)卵巢细胞株CO细胞分离培养大口黑鲈鱼弹状病毒,并用于佐剂灭活疫苗的制备;MSRV佐剂灭活疫苗免疫蛋鸡,成功获得MRSV的卵黄抗体,效价为1∶256,稀释度为1∶64时的中和病毒的作用最为明显,中和作用率达38.29%以上,病毒核酸拷贝数明显降低。同时,还可上调细胞内凝集素的表达。研究表明,特异性卵黄抗体对大口黑鲈弹状病毒具有明显的中和效果,为后续卵黄抗体作为免疫制剂的应用奠定了基础。
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| 关 键 词： | 大口黑鲈 弹状病毒 卵黄抗体 病毒中和 |
| 收稿时间： | 2019/7/4 0:00:00 |
| 修稿时间： | 2019/7/30 0:00:00 |
Isolation and Egg-Yolk Antibody Preparation of Micropterus salmonides Rhabdovirus |
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| Authors: | YUAN Xuemei LV Sunjian SHI Weid HANG Xiaoying LIU Li WU Yinglei |
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| Institution: | 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 |
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| Abstract: | The largemouth bass Micropterus salmoides has been widely cultured in China due to its considerable economic value. Recently, the species suffered from serious M. salmoides rhabdovirus(MSRV) disease during the juvenile phase, particularly in aquaculture conditions. In this study, a strain of M. salmoides rhabdovirus isolated from dying juvenile fish was cultured in a grass carp ovary(CO) cell line. To take advantage of passive immunity, we explored anti-MSRV immunoglobin Y(IgY) by discontinuously immunization on laying hens with a formalin-killed vaccine of MSRV. The antibody was purified by saturated ammonium sulfate, and displayed virus resistance with an ELISA antibody titer of 1∶256 detected. The bioactivity of anti-MSRV IgY was tested simultaneously after being added to a CO cell line incubated with MSRV. This antibody showed antiviral activity, with 38.29% of neutralizing effectiveness detected at a dilution of 1∶64. The RNA copies of MSRV in the test group were clearly fewer in number compared with those of members of a control group. By the florescent real-time quantitative PCR, the intracellular lectin experienced significantly up-regulated pick-up to nearly 10-fold at 72 h compared with the control group. In conclusion, we successfully isolated a strain of MRSV and cultured it in a CO cell line. Anti-MSRV IgY prepared with vaccinations of inactive virus displayed antiviral activity in this study, laying a foundation for the application of yolk antibodies as an immune preparation. |
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| Keywords: | Micropterus salmoides Rhabdovirus Egg-yolk antibody Virus neutralization |
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