【作者】 任秉新；

【导师】 樊廷俊；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2009， 博士

【摘要】 大菱鲆(Scophthalmus maximus)在分类学上属硬骨鱼纲(Osteichthyes)、鲽形目(Pleuronectiformes)、鲽亚目(Pleuronectoidei)、鲆科(Bothidae)、菱鲆属(Scophthalmus),为原产于欧洲的名贵经济鱼类,英文名为turbot,在我国俗称“多宝鱼”,是重要的海水经济增养殖鱼类之一。近年来,由于环境污染的日益加剧和养殖业的过度膨胀,各种传染性疾病尤其是病毒病开始广泛传播,病毒性疾病危害最大,并且极难防治。大菱鲆红体病是近年来对大菱鲆养殖业危害较大的一种病毒性疾病,其病原为大菱鲆红体病虹彩病毒(Turbot rddish body iridovirus,TRBIV)。大菱鲆死亡率逐年上升,给大菱鲆养殖业带来了巨大的经济损失。从根本上解决和预防大菱鲆病毒病的关键是要查清病毒的感染途径、感染机理以及研制病毒疫苗对大菱鲆进行免疫预防,而建立新的大菱鲆细胞系是进行病毒研究的理想体外研究体系和制备病毒疫苗的有效方法。本文采用胰蛋白酶、透明质酸酶和Ⅱ型胶原酶消化法成功启动了大菱鲆肝组织的原代培养,原代培养启动一周后,组织块周围即有大量的细胞迁出,形成明显的生长晕,30天后便可长成细胞单层。通过比较DMEM/F-12和L-15两种培养液,发现DMEM/F-12更适合大菱鲆肝细胞的体外培养。确定了大菱鲆肝细胞的最适培养体系为:20℃下,pH值7.0,添加50μg/mL羧甲基壳多糖、50μg/mL N-乙酰葡萄糖盐酸盐、10ng/mL表皮生长因子(EGF)、10 ng/mL碱性成纤维细胞生长因子(bFGF)和20%胎牛血清的DMEM/F-12培养液。经继代培养后,已传至32代。病毒感染体外培养细胞引起的细胞病变效应(cytopathic effects, CPE),是病毒在细胞内增殖的一个重要检测指标。本文利用TRBIV的敏感性细胞牙鲆腮细胞(Flounder gill,FG)为繁殖体系,以CPE为判断指标,对分离制备的TRBIV进行了鉴定,并测定其血凝效价作为病毒含量的标准,同时进行了体外最适繁殖条件如病毒接种量,吸附时间及细胞培养条件等的研究。结果表明,病毒接种量、病毒吸附时间、细胞培养条件均对TRBIV的体外繁殖产生明显的影响,如下繁殖条件可使病毒的繁殖速度最快:0.2 mL病毒液(血凝效价为1:64)接种于FG细胞,进行2h吸附后补加10%胎牛血清-MEM培养液,置22oC培养。另外还发现,病毒的感染力均受到病毒的收获时间和病毒液冻融次数的显著影响,在最佳繁殖条件下,收获在细胞中繁殖至第4天的病毒,且只冻融1次,可使其保持最强的感染活力,反复冻融会使病毒的感染活力显著降低。TRBIV的繁殖及其收获条件的确定,为后续大菱鲆病毒疫苗的研制奠定了基础。在TRBIV的体外最适繁殖条件下,对TRBIV进行了大量增殖,利用传统的甲醛灭活法制备TRBIV灭活疫苗,加入终浓度为0.1%福尔马林,于37℃进行灭活处理48小时,即获得灭活TRBIV疫苗原液,置4℃保存,待使用时再行稀释并加入终浓度为5mg/mL的Al(OH)3佐剂,制成灭活TRBIV疫苗使用液。对TRBIV疫苗的安全性检测结果显示,灭活TRBIV疫苗液不会引起FG细胞病变,也不会造成健康正常大菱鲆染病,证明所得病毒疫苗已被有效灭活,具有高度安全性,可用于大菱鲆的预防免疫。TRBIV疫苗对大菱鲆的免疫保护力检测结果证明,实验室内注射灭活TRBIV疫苗对10尾大菱鲆的免疫保护率为90%,养殖场内浸泡灭活TRBIV疫苗对两个养殖场中50万尾和200万尾大菱鲆的免疫保护率分别为83%和85%,证明所得灭活TRBIV疫苗对大菱鲆具有极高的免疫保护率;血清抗体检测结果显示,TRBIV疫苗接种8天后,大菱鲆体内血清的血凝抑制效价为1∶20,表明TRBIV疫苗诱导大菱鲆产生了抗TRBIV的抗体,可显著增强抗TRBIV感染的免疫力。更多还原

【Abstract】 Turbot, Scophthalmus maximus, has been one of important farmed fish species in coastal areas of northern China since it was introduced from Europe. However, epizootic disease of farmed turbots in China occurred recently because of intensive aquaculture with high density stocking and improper management. The pathogens were assumed to be parasite, bacteria, or virus. A recent study survey of farmed turbot diseases showed that“turbot reddish body syndrome”(RBS) was found in both juveniles and adults which was caused by a novel virus, turbot reddish body iridovirus (TRBIV). High mortalities of farmed turbot due to RBS diseases led to great economic loss. The best way for prevention is to best understand TRBIV infection, characterization and vaccination of TRBIV. The establishment of healthy and sensitive fish cell line is essential for isolation, identification and characterization of infectious viruses from fish.To initiate the primary culture of liver cells successfully, turbot liver tissues were digested with trypsin (0.25%), hyaluronidase (0.5%) and Type II callagenase (0.2%). The experiment of using different mediums DMEM/F-12 and Leibovitz-15 for liver cell culture was concluded that the optimal medium was DMEM/F-12 which can supply liver cells better growth and division. Primary culture of this cell type was conducted at pH 7.0, 20℃using DMEM/F-12 medium supplemented with antibiotics, 20% filtered bovine serum (FBS), basic fibroblast growth factor (bFGF, 10 ng/mL), epidermal growth factors (EGF, 10ng/mL), N-Acetyl-D-glucosamine (50μg/mL), and carboxymethyl-chitosan (50μg/mL). The emergence of new cell growth began from the edges of seeded tissue explants and was observed one week later after tissue explanting. The cells grew quite fast, and appeared uniformly fibroblast-like morphology. Thirty days later, the cells grew into confluence in 25 cm2 tissue culture flasks. When the TF cell grew into monolayer, they were subcultured via routine trypsin-digestion method. The TF cells have been subcultured to passage 32.In this study, TRBIV was separated from the diseased turbot with reddish body syndrome. By using cytopathic effect (CPE) in the form of cell rounding as a judgement index, monolayers cells of Flouder Gill (FG) cell line was used for TRBIV culture and the optimal propagation condition of TRBIV was studied. It was found that the amount of inoculated virus, adsorption time of virus and FG cell culture condition all had great effects on the in vitro propagation of TRBIV. And TRBIV propagated very fast when 0.2 mL virus solution was inoculated, adsorpted for 2 h, and virus inoculated FG cells was cultured in 10% bovine calf serum supplemented MEM medium at 22 oC. It was also found that the harvest time of propagated viruses and times of freeze-thaw of the harvested virus solution had enormous effects on the infective ability of TRBIV. The propagated TRBIV harvested on the 4th days after inoculation and freeze-thawed only once had highest infective ability. The determination of the optimal propagation condition of TRBIV will lay solid foundation for exploiture of turbot virus vaccine.TRBIV greatly propagated under the optimal condition studied before was killed at 37℃for 48h with final concentration of 0.1% formalin to be used as inactivated vaccine for healthy turbot in this paper. The inactivated TRBIV vaccine was stored at 4℃, and used with Al(OH)3 of 5mg/mL after proper dilution. The detection of successful inactivation by formalin was performed to assure the TRBIV vaccine is safe for turbot and won’t cause fish infected. And the dectection results showed that inactivated TRBIV vaccine could’t infect FG cells and healthy turbot any more. Then healthy turbots were vaccinated with inactivated TRBIV vaccine via muscle injection in indoor culture and immersion in farmed fishpond culture, in the meanwhile, the control groups were inoculated with 0.9% salt water instead of TRBIV vaccine. The results showed that the turbot group vaccinated with TRBIV vaccine has higher survival, and the relative percentage survival is 90% in indoor culture of 10 turbots, while about 83% and 85% in two farmed fishpond culture respectively of 500,000 and 2,000,000 turbots. Moreover specific antibody titres could be detected of 1∶20 at 8 days after injecetion immunization. All of these results concluded that the inactivated TRBIV vaccine was effective for protecting turbots against infection of TRBIV.更多还原