鳜脑组织细胞系的建立及其病毒敏感性研究

鱼类细胞是开展鱼类病毒分离鉴定、功能基因分析以及生物制品制备等研究的重要物质基础。鳜(Siniperca chuatsi)是深受养殖者和消费者欢迎的养殖品种。随着鳜鱼养殖产量的逐年增加,其病害问题尤其是病毒病问题也日趋严重,但是,可用于鳜鱼病毒分离和基因功能分析的鳜细胞系缺乏。本研究采用组织块消化法,对来源鳜脑组织的细胞进行原代培养,建立了鳜脑组织细胞系,命名为MFB。MFB细胞在28℃含10%胎牛血清的L-15中已稳定传代超过70次,第25代鳜脑组织细胞的染色体众数为56。采用免疫荧光细胞化学技术(β-tubulin和Neu-N)鉴定MFB细胞的神经元纯度,结果显示,培养的MFB细胞为神经元类细胞。病毒敏感性实验结果显示,鳜蛙虹彩病毒(MFRaIV)、大口黑鲈蛙虹彩病毒(LMBRaIV)和大鲵虹彩病毒(GSIV)均可在MFB细胞中产生典型细胞病变效应,病毒滴度分别为108.68±0.12、108.36±0.15、1010.15±1.85 TCID50/mL。使用脂质体Lipofectamine®2000将pEGFP-N1转入MFB细胞,转染效率可达20%。本研究建立的鳜脑组织细胞系不仅对多种蛙虹彩病毒敏感,而且转染质粒效率较高,为鳜病毒性病原的分离及基因功能研究奠定了前期基础。     

三种脂质体介导的花鲈胚胎干细胞转化效率的比较

通过3种脂质体(genejammer, genejuice和metafectene)介导绿色荧光蛋白基因转化花鲈胚胎干细胞，探讨不同脂质体介导的花鲈胚胎干细胞（LJES1）的转化效率，以及影响外源基因转化效率的因素，如细胞接种时间、初始接种密度、DNA和脂质体用量以及它们之间的比例等。实验发现，genejammer转化LJES1细胞效率最高，适合于该细胞系的转化。对于同一种脂质体，DNA与脂质体用量的比例对于绿色荧光蛋白基因转化LJES1最为重要，DNA与genejammer的比例为1∶6时，转化效率最高，为27.3%；DNA与genejuice的比例为1∶4时，转化效率最高，为12.1%；而DNA与metafectene的比例为1∶3时，转化效率达到最高，为5.3%。随着DNA、脂质体量的增加，转化效率有所提高，但达到一定的用量后，转化效率反而下降。     

鳜SIRT3蛋白在传染性脾肾坏死病毒感染中的作用

沉默交配型信息调节因子 2同源蛋白 3 (silent mating type information regulation 2 homolog 3, SIRT3)是一种去乙酰化酶, 可调节线粒体氧化代谢。为探讨鳜(Siniperca chuatsi) SIRT3 在传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus, ISKNV)感染中的作用, 本研究克隆分析了鳜 SIRT3 基因, 采用 qRT-PCR 和 western blotting 方法检测了病毒感染后 SIRT3 基因的表达, 并探讨了 SIRT3 对细胞代谢酶基因的表达和病毒增殖的影响。结果显示, 鳜 SIRT3 基因的开放阅读框全长 1350 bp, 编码 449 个氨基酸, 在鳜各组织均表达, 其中后肾中表达量最高, 脾脏中表达量较低; ISKNV 感染鳜脑细胞系和鱼体后, SIRT3 表达量均显著升高; 使用 SIRT3 抑制剂 3-TYP (10 μmol/L)和 siRNA 处理细胞后, 谷氨酰胺酶(glutaminase, GLS)和谷氨酸脱氢酶(glutamate dehydrogenase, GDH)的表达量以及 ISKNV 产量均显著降低; 使用 SIRT3 激动剂 honokiol (7 μmol/L)处理细胞后, 代谢酶表达量和 ISKNV 产量均显著升高。结果表明, ISKNV 感染可通过 SIRT3 诱导宿主细胞谷氨酰胺代谢重编程, 进而促进自身增殖。     

大菱鲆受精卵显微注射技术的建立

本研究将100~300 pg含有肌肉特异表达启动子和绿色荧光蛋白(Green fluorescent protein,GFP)基因的重组质粒(smyd1:gfp)显微注射到大菱鲆(Scophthalmus maximus)受精卵动物极细胞中,通过细心培育,成功孵化出鱼苗约120尾。统计分析显示,显微注射后,大菱鲆胚胎存活率为4.8%。利用荧光显微镜观察大菱鲆胚胎及仔鱼,只在注射smyd1:gfp质粒的胚胎及仔鱼的肌肉中发现有绿色荧光。通过进一步PCR扩增检测,在注射的大菱鲆胚胎及仔鱼DNA中扩增出了GFP特异片段,大小约为340 bp。研究表明,本研究成功建立了大菱鲆显微注射技术,可为大菱鲆基因功能研究和遗传育种奠定基础。     

翘嘴鳜学习记忆相关基因c-fos对食欲基因的调控

学习记忆在动物获得新的觅食技巧和食物偏好方面扮演着重要的角色。为评估学习记忆对翘嘴鳜(Siniperca chuatsi)摄食调控的影响, 构建翘嘴鳜学习记忆相关基因 c-fos 及 zif268 荧光过表达载体, 并以 pcDNA3.1-EGFP 载体质粒中的绿色荧光蛋白基因为报道基因进行电转实验, 优化电压、脉冲时间、质粒浓度和电击次数等电转条件, 确立最佳条件。利用电击的方式转染翘嘴鳜脑细胞, 检测食欲基因 mch、agrp、pomc、npy 表达变化。结果显示, 成功构建了学习记忆过表达载体 pcDNA3.1-c-fos-EGFP、pcDNA3.1-zif268-EGFP。翘嘴鳜脑细胞电转染的最适条件: 细胞密度 1×106 /mL、电压 240 V、脉冲时间 5 ms、质粒 6 μg、电击 1 次、转染 48 h, 可见大部分荧光蛋白表达, 转染率较高, 表明 pcDNA3.1-EGFP 在体外成功转染翘嘴鳜脑细胞。过表达 c-fos 基因后, 食欲相关基因 pomc 和 npy mRNA 水平均显著上升。而过表达 zif268 基因后, 并未发现食欲基因有差异表达。以上结果说明, 翘嘴鳜学习记忆相关基因 c-fos 的表达影响了食欲相关基因 pomc 和 npy 的表达。     

鳜弹状病毒糖蛋白在杆状病毒表达系统中的表达与鉴定

近年来,鳜弹状病毒引起的病害频发,该病毒传播快,致病性强,是引起鳜病害的主要病原之一。鳜弹状病毒基因组编码5个结构蛋白,其中糖蛋白G是病毒表面的主要抗原,为实现该抗原蛋白的大量表达,利用分子克隆技术将鳜弹状病毒糖蛋白G片段插入杆状病毒穿梭载体pFastBac1中转化至大肠杆菌DH5α感受态细胞后,筛选阳性克隆获得重组转移载体pFastBac-G,再将其转化至大肠杆菌DH10Bac感受态细胞,经蓝白斑与抗性筛选后获取重组杆粒rBacmid-G,随后利用脂质体转染法将重组杆粒转染Sf9昆虫细胞制备重组杆状病毒。将获取到的P3代重组病毒感染Sf9昆虫细胞进行重组蛋白的SDS-PAGE检测,成功表达后利用镍离子亲和层析柱纯化目的蛋白,随后对重组蛋白进行Western blot鉴定,结果显示在大于50 ku处有一条特异性条带,而对照组无条带,表明制备的抗原能与抗体特异性结合。结果表明,鳜弹状病毒糖蛋白G在杆状病毒表达系统成功表达。本试验结果可为鳜弹状病毒糖蛋白G疫苗的研发和大规模生产提供技术支撑。     

草鱼呼肠孤病毒衣壳蛋白VP7基因真核表达载体pCI-VP7的构建及鉴定

将编码草鱼呼肠孤病毒(grass carp reovirus,GCRV)主要衣壳蛋白VP70.9kb的基因片段连接至克隆载体pMD19-T中,筛选阳性克隆并测序,经检测为正确序列后,再将目的片段克隆入真核表达载体pCI,筛选得到阳性重组质粒pCI-VP7。然后构建pCI-VP-GFP重组表达质粒(即GFP基因与VP7的一段上游基因融合表达),用PCR及酶切方法鉴定克隆的正确性。并用脂质体法将其转染入真核细胞COS-1和CIK进行瞬时表达,荧光显微镜观察及RT-PCR特异性检测。结果表明,GFP基因与VP7的一段上游基因被成功转染到COS-1和CIK细胞,并得到了很好的表达。进而证明pCI-VP7可以成功的表达,为GCRV基因疫苗的研制提供了实验资料。     

斑石鲷肾脏细胞系的建立、特征分析及其免疫相关基因的表达分析

斑石鲷(Oplegnathus punctatus)是我国重要的海水养殖鱼类, 本研究以斑石鲷肾脏组织为材料, 建立了斑石鲷肾脏组织细胞系(O. punctatus kidney cell line, OPK)。斑石鲷肾脏细胞系生长旺盛, 目前已成功传至 34 代。本研究检测了不同 FBS 浓度对斑石鲷肾脏细胞生长的影响, 结果表明最适生长 FBS 浓度为 20%。将 OPK 细胞液氮冷冻复苏后, 细胞具有活性, 可正常生长和传代。核型分析结果显示, 第 24 代斑石鲷肾脏细胞系核型为正常二倍体核型(2n=2sm+46t)。将 Cy3-siRNA 转染到 OPK 细胞后, 可以成功表达荧光。对斑石鲷 OPK 细胞提取 DNA, 用斑石鲷线粒体色素细胞 C 氧化酶 I (CO I)基因进行检测, 结果表明该细胞系来源于斑石鲷。斑石鲷肾脏细胞系细胞在受到脂多糖(LPS)和聚肌胞苷酸(poly I∶C)刺激后能产生免疫反应, 免疫相关基因 IκB、IL-1β、IL-8 和 IRF3 的表达水平发生显著变化。本研究成功建立了斑石鲷肾脏细胞系, 可运用于斑石鲷基因功能分析、细胞遗传学、致病性细菌和病毒感染机制等研究, 可为斑石鲷的基础研究和细胞工程育种等提供重要的基础材料。     

草鱼jam-a分子在胚胎幼鱼期及受GCRV感染PSF细胞的表达分析

草鱼呼肠孤病毒(Grass carp reovirus, GCRV)可引发草鱼(Ctenopharyngodon idellus)出血病,导致高死亡率。草鱼吻端成纤维细胞(Grass carp snout fibroblast cells, PSF)是GCRV的敏感细胞系。JAM-A (Junctional adhesion molecule A)为免疫球蛋白超家族成员,是多种病毒的细胞受体。本研究在前期克隆到草鱼3种jam-a基因,命名为gcjam-a1,gcjam-a2和gcjam-a3。在获取ORF序列的基础上,利用qRT-PCR分析了3种gcjam-a在草鱼胚胎及幼鱼不同发育时期及PSF细胞中受GCRV(GD108株)感染前后的表达模式。结果显示,检测的13个胚胎及幼鱼发育时期中,gcjam-a1在未受精卵中高表达,在受精卵至出膜前的胚胎表达水平均较低;从出膜后1~3 d表达量开始上升;出膜6~15 d均呈高水平表达。gcjam-a2与gcjam-a3在草鱼胚胎及幼鱼发育各阶段表达水平较低。在无病毒感染的PSF细胞中,gcjam-a只有少量表达。受GCRV-GD108感染后,病毒S7基因在PSF细胞中的拷贝数随时间呈显著上调趋势,gcjam-a的表达量也有不同程度的上调,mRNA上调水平为gcjam-a1>gcjam-a2>gcjam-a3。本研究证实了3种gcjam-a基因在PSF细胞中的表达均与GCRV-GD108感染相关,其中,gcjam-a1的表达水平受GCRV-GD108感染影响最大,同时,它在孵化出膜后表达上升,推测它可能与病毒的感染更相关。gcjam-a1可作为下一步GCRV与宿主互作研究中的候选分子。     

稳定表达草鱼LamR鱼类细胞的建立及对基因Ⅱ型GCRV增殖的影响

通过构建草鱼LamR(Ctenopharyngodon idella LamR,CiLamR)真核表达质粒,转染GCRV非敏感细胞系鲤鱼上皮细胞(EPC)和敏感细胞系草鱼肾细胞(CIK),利用G418筛选获得稳定表达CiLamR的细胞,经Western Blot验证CiLamR稳定表达细胞系的构建。采用Ⅱ型GCRV分别接毒CiLamR稳定表达EPC和CIK,检测接毒后Ⅱ型GCRV拷贝数差异,研究草鱼37 kDa/67 kDa层粘连蛋白受体(37 kDa/67 kDa laminin receptor, LamR)对Ⅱ型GCRV增殖的影响。结果显示:经PCR扩增获得927 bp CiLamR完整开放阅读框(ORF),并成功克隆进入真核表达质粒pEYFP-Mem,获得pEYFP-Mem-CiLamR重组质粒,转染并经过G418筛选后获得约80%阳性荧光的细胞。Western Blot证实稳定表达CiLamR的EPC和CIK细胞系构建成功。以不同初始浓度接种EPC-pEYFP-Mem、EPC-pEYFP-Mem-CiLamR细胞均未检测到Ⅱ型GCRV增殖,CIK-pEYFP-Mem、CIK-pEYFP-Mem-CiLamR可检测到病毒增殖,但是增殖量无明显差异。结果表明:CiLamR蛋白对基因Ⅱ型GCRV增殖无明显影响。     

Meiotic gynogenesis with heterologous sperm in the mandarin fish Siniperca chuatsi and evidence for female homogamety

The mandarin fish Siniperca chuatsi is a historically important aquaculture species in China and exhibits sexually dimorphic growth. However, sex determination of this fish remains unclear so far. In this study, we induced meiotic gynogenesis in S. chuatsi using irradiated heterologous sperm from spotted mandarin fish (Siniperca scherzeri) to uncover its mechanism of sex determination. Up to 7.52% diploid progeny were obtained among three gynogenetic families in this study. Molecular analysis of female and male donors and sampled young gynogens by seven microsatellite loci further confirmed no genetic contributions from the ‘father’ S. scherzeri. After 8 months of culture, external morphology of adult fish showed that all gynogens were cloned from their mothers. Gonads of the gynogenetic progeny were examined by histological observations and the sexing results showed that they were almost 100% females, strongly supporting an assumption of female homogamety in mandarin fish. By this study, we obtained pure lines of S. chuatsi and elucidated its genetic mechanism of sex determination, providing a basis for possible sex control breeding in this species.     

鳜视觉和侧线感觉调控捕食行为的动态观察

鳜（Siniperca chuatsi）具有独特的捕食习性。为研究鳜捕食行为相关主要感觉——视觉和侧线感觉调控的捕食行为特征,利用高速摄像与感官抑制技术对鳜的独特捕食行为进行了分析,将实验鳜分为4组：G_C（对照组,视觉与侧线感觉均具备）,G_V（只有视觉）,G_L（只有侧线感觉）和G_DS（视觉、侧线感觉均不具备）4组。对各组鳜投喂饵料鱼,并利用高速摄像技术对单位时间内鳜的捕食行为进行动态观察与分析。结果表明,鳜在捕食行为中表现出5种捕食模式：直接攻击型、跟踪-弹射型、跟踪-偏移型、弹射型、偏移型,其中,直接攻击型、跟踪-弹射型、弹射型捕食模式主要由视觉控制,跟踪-偏移型、偏移型主要由侧线感觉控制。在视觉、侧线感觉均具备时优先利用视觉捕食,捕食行为趋于简化,相反,鳜仅利用侧线感觉捕食时,捕食行为变得复杂而多样;鳜在捕食时所表现出的各种捕食行为模式是其具有独特摄食习性的重要原因。上述研究为解读鳜特殊捕食行为的形成机制提供了重要资料。     

Ammonia nitrogen excretion in Mandarin Fish (Siniperca chuatsi) and Grass Carp (Ctenopharyngodon idellus) fed practical diets: the effects of water temperature

To understand the actual production of fish culture about the utilization of dietary protein and excreta impact on the environment between mandarin fish (Siniperca chuatsi) and grass carp (Ctenopharyngodon idellus), the study to investigate the effect of temperatures (19 ± 0.5°C, 24 ± 0.5°C and 29 ± 0.5°C) on ammonia‐N excretion in mandarin fish and grass carp under fed and fasted states was conducted. These two species were fed a practical diet containing 325.2 g kg^?1 crude protein at 3% body weight per day. The ammonia‐N excretion rate was significantly increased when temperature increased from 19 to 29°C, and a linear relationship between ammonia‐N excretion rate and temperature. The maximum ammonia‐N excretion levels of mandarin fish and grass carp were observed at 4–8 h and 2–4 h after feeding, respectively, and the minimum values for both species were observed at 24 h after feeding. Under the feeding condition, mandarin fish had a lower ammonia‐N excretion level compared to grass carp at 24°C and 29°C. The average amount of ammonia‐N excreted by mandarin fish at 24 h is significantly higher than grass carp under fasting conditions, except 19 ± 0.5°C. These results indicated that mandarin fish might make better use of protein at higher temperature than grass carp when fed practical diets in commercial production. These results of this study suggested that mandarin fish had a lower ammonia‐N excretion level compared with grass carp, making a less contribution to environmental loading in an intensive fish culture.     

Disease resistance and immune‐relevant gene expression in golden mandarin fish,Siniperca scherzeri Steindachner,infected with infectious spleen and kidney necrosis virus‐like agent

Infectious spleen and kidney necrosis virus (ISKNV), family Iridoviridae, genus Megalocytivirus, may cause high mortality rates such as those seen in mandarin fish, Siniperca chuatsi. ISKNV has attracted much attention due to the possible environmental threat and economic losses it poses on both cultured and wild populations. We have investigated the pathogenicity of ISKNV‐like agent Megalocytivirus, isolated from infected pearl gourami, in golden mandarin fish, Siniperca scherzeri – a member of the Percichthyidae family – and in another Percichthyidae species, S. chuatsi. Fish were challenged with four different doses of ISKNV‐like agent Megalocytivirus (1, 10, 100 or 1000 μg per fish) over a 30‐day period, and cumulative fish mortalities were calculated for each group. No significant mortality was observed for fish challenged with the lowest dose (1 μg per fish) relative to a control group. However, all other challenged groups showed 100% mortality over a 30‐day period in proportion to the challenge dose. Quantitative real‐time PCR was performed to measure mRNA expression levels for six immune‐related genes in golden mandarin fish following ISKNV‐like agent challenge. mRNA expression levels for IRF1, Mx, viperin and interleukin 8 significantly increased, while mRNA levels for IRF2 and IRF7 remained constant or declined during the challenge period.     

大黄鱼肿大细胞病毒不同毒株的细胞培养及主要衣壳蛋白基因比较

为建立大黄鱼肿大细胞病毒的培养方法,明确其分类地位,用肿大细胞病毒检测呈阳性的大黄鱼幼鱼病料 (FD201807和SA201808)肾组织匀浆液感染鳜仔鱼细胞系 (mandarin fish fry cell line-1,MFF-1)并连续传代,从病料组织匀浆液和细胞冻融液中提取病毒DNA,克隆病毒主要衣壳蛋白基因 (mcp),测序后与NCBI GenBank中的虹彩病毒科肿大细胞病毒属病毒mcp以及2018—2020年所检出的15株大黄鱼肿大细胞病毒mcp进行比对分析。结果显示,病毒传至第4代才可引起MFF-1细胞病变,细胞病变的主要特征为细胞脱壁、变圆、折光度增强;感染时间越长脱壁细胞越多,同时培养液中的颗粒增加;透射电镜下可见感染细胞的细胞质散在大小为130~150 nm的六边形病毒粒子和空壳。感染细胞的病变周期随传代代次的增加而缩短,第15代次的FD201807株感染细胞80%细胞病变的时间为3 d,第15代次的SA201808株感染细胞80%细胞病变的时间为7~8 d。mcp序列比对和聚类分析发现,SA201808株与FD201807株的mcp序列存在21个碱基差异,二者的mcp序列分别与大黄鱼虹彩病毒(large yellow croaker iridovirus, LYCIV) LYCIV-Zhoushan (GenBank: MW139932.1)和花鲈虹彩病毒 (Lateolabrax maculatus iridovirus, LMIV) (GenBank: MH577517.1)相近。15株从大黄鱼病料检出的肿大细胞病毒中,12株的mcp序列与SA201808株聚类;3株与FD201807聚类。本研究利用MFF-1细胞系分离培养了大黄鱼肿大细胞病毒,揭示了大黄鱼肿大细胞病毒存在差异,为更好地了解大黄鱼肿大细胞病毒提供了数据参考。     

Effects of live and artificial feeds on the growth,digestion, immunity and intestinal microflora of mandarin fish hybrid (Siniperca chuatsi ♀ × Siniperca scherzeri ♂)

How to acclimate mandarin fish to eat artificial feeds has been always a challenge for researchers. The mandarin fish after hybridization (Siniperca chuatsi ♀ × Siniperca scherzeri ♂) could be fed artificial feeds which solved the problem to some extent. However, the growth performance, digestibility, immunity and intestinal microflora of mandarin fish hybrid fed artificial feeds need further study. One hundred and twenty fish of similar size (average weight, 19.5 ± 0.9 g) were randomly stocked into six 250‐L aquariums and separately fed live baits (the control) or artificial feeds in triplicate for 70 days. The weight gain and special growth rate of fish fed artificial feeds were significantly lower than those of fish fed live baits (P < 0.05). The protease activities of stomach, liver and intestine in fish fed live baits were all significantly higher than those in fish fed artificial feeds. The activities of catalase and lysozyme, the content of glutathione in serum of fish fed live baits were all significantly higher than those in fish fed artificial feeds. However, the content of malondialdehyde in liver of fish fed artificial feeds was significantly higher than that in fish fed live baits. The dominant bacteria in both groups were Proteobacteria, Bacteroidetes, Planctomycetes, Cyanobacteria, Verrucomicrobia. However, live baits greatly affected the amount of beneficial and harmful bacteria of intestine in mandarin fish hybrid and broke the balance of intestinal flora.     

Liposome‐mediated messenger RNA: An alternative for fish cell transfection in culture

Liposome‐mediated plasmid DNA (pDNA) transfection is a relatively efficient transfection method that depends highly on actively dividing cells. However, numerous fish cells are mitotically inactive under in vitro culture conditions, thus limiting the application of liposome‐mediated pDNA transfection. Liposome‐mediated mRNA transfection is an attractive alternative in post‐mitotic cells because it eliminates the transfer of genes into the nucleus and therefore is independent of cell proliferation. In this study, pDNA is compared with mRNA encoding biomarker proteins (enhanced green fluorescent protein [eGFP] and luciferase) mediated by cationic liposome in zebrafish embryo fibroblast cell line (ZF4), with or without proliferation restriction, and in mitotically inactive turbot liver primary cells. The results indicated that mRNA encoding eGFP exerted much higher transfection efficiency than pDNA in ZF4 cells. Moreover, mRNA encoding eGFP was detected and disappeared sooner than pDNA. Chemiluminescence of the biomarker protein luciferase showed that liposome‐mediated mRNA transfection translated about threefold more protein without proliferation restriction and about tenfold more protein with proliferation restriction than pDNA transfection. In turbot liver cells, mRNA induced higher transfection efficiency and greater luciferase protein expression than pDNA. Greater PPARα expression and expression of downstream target genes were induced by mRNA rather than by pDNA transfection. In conclusion, cationic liposome‐mediated mRNA is an alternative for fish cell transfection due to higher transfection efficiency and protein expression levels and faster translation onset.     

Establishment of a cell line from the kidney of black carp and its susceptibility to spring viremia of carp virus

In this study, we established and characterized a cell line derived from the kidney of black carp (Mylopharyngodon piceus), which is an important freshwater aquaculture species. The cell line was designated as MPK and subcultured for more than 70 passages in DMEM medium containing 10% fetal bovine serum (FBS) at 28°C. MPK had a modal diploid chromosome number of 48. Moreover, a transient MPK transfection efficiency was up to 18% using a green fluorescent protein plasmid by a modified electroporation. In addition, the MPK cells showed susceptibility to spring viremia of carp virus (SVCV), as demonstrated by the presence of severe cytopathic effects (CPEs) and increased viral RNA. Unexpectedly, the MPK cells expressed pluripotency‐associated genes such as nanog, oct4 and vasa, indicating that these are possibly adult stem cells. Taken together, we have established a stable cell line from kidney that may potentially be utilized as an in vitro platform for genetic modifications and host–pathogen analysis in black carp.