草鱼呼肠孤病毒微载体培养技术与灭活疫苗浸泡免疫效果分析-临床兽医学专业论文.docxVIP

上海海 上海海洋大学硕士学位论文 II II 吞噬活性、血清超氧化物歧化酶（SOD）活性和血清抗体效价，同时提取免疫鱼 和对照鱼肝脏、脾脏和头肾 3 种组织中的总 RNA 并逆转录成 cDNA，采用荧光定 量 PCR 技术对不同组织中免疫相关基因 IgM 和 Mx 基因的表达进行分析。结果表 明，浸泡免疫可以诱导草鱼血液中红细胞和白细胞数量的增加，显著提高血液中 白细胞的吞噬活性和血清中 SOD 活力，增强草鱼的非特异性免疫功能；同时免疫 鱼体内血清中的抗体效价也显著高于对照组，且在 21 d 时抗体效价达到峰值，IgM 和 Mx 基因在三种组织中的表达均呈现不同程度的上调，IgM 基因在肝脏中的表达 与对照组之间未出现显著性差异，在脾脏和头肾中的表达都显著高于对照组，分 别在 14 d 和 21 d 达到峰值。Mx 基因在免疫鱼肝脏中的表达于免疫后 7、14 d 显著 高于对照组，在脾脏和头肾中的表达于免疫后都显著高于对照组，且在免疫后 14 d 达到峰值。IgM 和 Mx 基因在脾脏和头肾中都有较大的表达量，而在肝脏中的表达 量较小。免疫 28 d 后攻毒试验结果表明免疫组的免疫保护率为 76%。浸泡免疫可 以通过草鱼血细胞增殖、提高白细胞吞噬活性、产生特异性抗体及提高免疫相关 基因的表达量等方式增强机体的免疫保护力。 关键词：草鱼出血病，肾脏组织细胞系（CIK），呼肠孤病毒，微载体培养，灭活 疫苗，浸泡免疫 III III Studies on the Cultivation of Grass Carp Reovirus by Microcarrier and the Immune Effect of Immersion with Killed Vaccine ABSTRACT Grass carp (Ctenopharyngodon idellus) is the major species of freshwater aquaculture in China, which has numerous advantages including rapid growth rate, various food sources and delicious taste, etc. Grass carp hemorrhage is the most severe infectious disease affecting grass carp fingerlings caused by grass carp reovirus (GCRV), and leads to huge economic loss in the industry of grass carp culture. The grass carp hemorrhage in a wide range of areas with a long period and and causes high mortality. At present, immunization and natural plant anti-virus drugs therapy are the major methods to prevent the disease. The GCRV propagation for inactivated vaccine preparation is mainly dependant on the adherent culture of CIK cells on bottles, which represents a limiting factor for the large-scale production of the vaccine. Currently, there are three major methods for fish vaccination, including intraperitoneal injection, immersion and oral immunization. Although the vaccination by immersion is a simple and convenient method, the mechanism of immune response and the immune efficacy are needed to be investigated in-depth. In this paper, the microcarrier culture technology of grass carp cell and reovirus, mechanism of immunization by imm