青鱼鳍条组织细胞系的建立及其生物学特性

采用组织块移植培养技术,对来源于青鱼(Mylopharyngodon piceus)的鳍条组织细胞进行原代培养,建立了青鱼鳍条组织细胞系,定名为BC-Fin。青鱼鳍条组织细胞为成纤维样细胞,已稳定传代培养50多代,其最适培养温度为25℃,最佳培养基为L-15,最适血清浓度为15%,在最适培养条件下,青鱼鳍条组织细胞的群体倍增时间为60.6 h。青鱼鳍条组织细胞液氮冷冻保藏6个月后,经台盼蓝染色,约(90.09±4.65)%的细胞具有细胞活性,复苏后的细胞生长旺盛。细胞染色体分析显示,第16代青鱼鳍条组织细胞的染色体数目为正常二倍体(2n=48),第41代细胞染色体众数为46。通过对离体培养细胞的线粒体中的16S rRNA基因进行特异性扩增,获得长度为320 bp的核酸片段,核酸序列比对分析结果表明,其与青鱼16S rRNA基因序列的一致性达98%,表明该细胞来源于青鱼。病毒敏感性试验结果显示,在感染草鱼呼肠孤病毒(GCRV)后BC-Fin细胞系可产生典型细胞病变效应,病毒滴度为105.33±0.21TCID50/m L,且PCR检测可检测出细胞培养的草鱼呼肠孤病毒,表明BCFin细胞系对草鱼呼肠孤病毒较敏感。     

镉污染对鲤鱼过氧化氢酶活性的影响

试验结果表明,试验剂量范围内,10 d 后Cd2+ 对鲤鱼的5种组织的过氧化氢酶(CAT)有不同程度的影响,其剂量-效应曲线为抛物线型,CAT活性始终表现为不同程度的诱导作用.当鲤鱼受到低质量浓度(0.00～0.6275 mg/L)Cd2+ 影响时,其5种组织的CAT活性表现为升高,当受到高质量浓度(0.6275～1.2550 mg/L)Cd2+ 影响时,其组织内CAT活性则表现为不同程度的降低,其中血液的敏感性最高,可作为灵敏指示镉污染的优先组织;其次肾脏中的CAT活性受镉离子的影响也很大,可能是因为肾脏是机体主要的排泄器官及肾小球过滤膜带负电荷的缘故.     

鲤疱疹病毒Ⅱ型的理化及生物学特性和超微形态发生

为了查明鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,Cy HV-2)的理化与生物学特性以及病毒在细胞内的超微形态发生过程,利用新建立的对Cy HV-2敏感的异育银鲫脑组织细胞系(Gi CB),对Cy HV-2的理化及生物学特性进行了详细研究,比较了不同来源鱼类细胞系对Cy HV-2感染的敏感性,并对体外培养细胞中Cy HV-2病毒粒子及其超微形态发生过程进行了电镜观察。结果显示,Cy HV-2对热、酸、碱、有机溶剂和冻融敏感;常用鱼类细胞系EPC、RTG-2、Koi-Fin、CIK、CCK、PF-Fin对Cy HV-2的感染不敏感,特异性巢式PCR检测盲传至第7代Cy HV-2细胞培养物,结果均为阴性;Cy HV-2在Gi CB细胞中的增殖动态研究结果表明:病毒感染细胞经过12 h的隐晦期,24 h开始进入对数生长期,96 h病毒滴度达到最高值(107.52±0.26 TCID50/m L),然后进入平台期;透射电子显微镜观察结果显示,Cy HV-2感染细胞可分为吸附与侵入、复制与装配、成熟与释放3个主要过程,病毒进入对数生长期后,被感染细胞内可见形态典型的疱疹病毒颗粒。     

镉诱导鲫肝细胞内Ca2+-ATP酶与金属硫蛋白的表达

研究了镉诱发鲫肝细胞相关的胞内游离钙离子变化,以及Ca²⁺-ATP酶及金属硫蛋白表达量的变化。试验分为对照组、5、10、15、20 μmol/L CdCl₂ 5个组。Ca²⁺用Fura-2/AM方法检测,试验后24 h用荧光倒置显微镜观察细胞内游离钙离子变化;分光光度法检测Ca²⁺-ATP酶;石墨炉—原子分光光度法检测了细胞内镉离子浓度;免疫酶联法(ELISA)检测了金属硫蛋白(MT)含量。结果显示,镉可导致细胞存活率下降,具有一定的毒性。镉离子引起胞内Ca²⁺荧光强度和Ca²⁺-ATP酶活性增加(P＜0.01)。随镉浓度升高,处理组Ca²⁺-ATP酶浓度活性分别是对照组的4.52、6.73、6.68、7.19、6.18倍;暴露24 h后各组细胞内镉离子均有上升,其中5 μmol/L组最高,达(2.045±0.322) μmol/L;各处理组金属硫蛋白(MT)含量增高(P＜0.01),且5 μmol/L低浓度组MT增幅最大,达17.15%。结果提示,镉诱导下细胞内Ca²⁺升高,MT表达量上升,且MT可螯合进入细胞内的镉离子,这种螯合可能是降低镉毒理作用的机制之一。     

丁香酚和MS-222对松江鲈的麻醉效果

采用静水试验方法，研究了不同质量浓度的两种常用渔用麻醉剂丁香酚(5、10、20、40、80、120 mg·L^-1)和MS-222(40、50、60、75、100、200 mg·L^-1)对体质量为(13.78±3.15) g的松江鲈(Trachidermus fasciatus)的麻醉效果。结果表明：1)随着两种麻醉剂浓度的升高，松江鲈进入麻醉状态的时间逐渐缩短，复苏时间不断延长；2)丁香酚与MS-222的最适麻醉质量浓度分别为40～80 mg·L^-1和60 mg·L^-1;3)在低浓度麻醉剂处理下，10 mg·L^-1丁香酚组中松江鲈的耗氧率显著低于对照组(P<0.05),表明10 mg·L^-1丁香酚可能是松江鲈的适宜运输浓度；4)对比成活率、进入麻醉状态的时间和复苏时间等指标，丁香酚更适合作为松江鲈的麻醉剂，在实际操作中，建议使用40～80 mg·L^-1丁香酚进行松江鲈的麻醉。     

凡纳滨对虾循环水养殖系统应用研究

以凡纳滨对虾(Litopenaeus vannamei)室内工厂化流水养殖(IIFA)为对照组,通过养殖场凡纳滨对虾循环水养殖(RAS)试验(85 d)比较不同养殖模式对凡纳滨对虾的生长性能、养殖水体水质影响,探究循环水养殖系统(RAS)的硝化效率变化。结果显示:RAS的凡纳滨对虾存活率(74.58%±1.74%)、饲料转化率(70.56%±3.82%)、产量(3.91±0.49 kg/m^3)显著高于IIFA的凡纳滨对虾存活率(66.90%±3.80%)、饲料转化率(67.14%±3.25%)、产量(3.47±0.42 kg/m^3)(P<0.05)。对虾RAS可以将养殖水体化学需氧量(COD)、氨氮(NH_4^+-N)和亚硝酸盐氮(NO_2^--N)质量浓度稳定在较低水平(5.92、0.60和1.14 mg/L);对照组的COD呈现上升趋势,最高升至15.37 mg/L,NH_4^+-N和NO_2^--N质量浓度在较大范围(0.20～2.90 mg/L和0.19～6.97 mg/L)内波动。然而,对虾RAS养殖水体NO_3^--N和总氮呈现逐渐上升的趋势,最高分别升至25.98和33.55 mg/L;对照组养殖水体NO_3^--N(0.94～2.85 mg/L)和总氮(5.95～14.01 mg/L)质量浓度变化则相对较小。对虾RAS对养殖水体硝化作用发挥着至关重要的作用,NH_4^+-N和NO_2^--N去除率分别为23.78%～91.43%和0～27.76%,NO_3^--N累积率则稳定在一定范围(0.57%～4.30%)。研究表明,对虾RAS的应用可有效控制凡纳滨对虾养殖水体关键水质指标,有利于对虾存活率的提高和养殖产量的增加。     

2株海洋细菌对镉的吸附作用

用高质量浓度镉培养液单独和联合培养2株海洋细菌J2和J6,3、7、10、14、17d后,采用原子吸收法分别测定培养液上清液和菌细胞中Cd2+的质量浓度,以确定2株菌对镉的吸附性能;通过测定菌体细胞内半胱氨酸脱巯基酶的活性,初步研究了2株菌对镉的转化作用机理。试验结果表明,2株菌均可将细胞外的Cd2+吸收到细胞内,在培养至第7d时,上清液中Cd2+的含量达到最低值,随着培养时间的增加逐渐趋于稳定,J2和J6菌体细胞中Cd2+的含量分别在培养到第10d和14d时达最大值,联合培养对镉的吸附作用优于单独培养;J2和J6在镉处理后细胞内的半胱氨酸脱巯基酶活性均显著增高,提示Cd2+的吸附与细胞内半胱氨酸脱巯基酶作用产生的S2-有关。     

紫外线诱变建立草鱼抗出血病病原病毒的AHZC88细胞株

通过UV对草鱼出血病病原病毒敏感的草鱼ZC 7901细胞株反覆诱变,获得了对出血病病原病毒具有抗性的AHZC 88细胞株。经生物学特性测定,AHZC 88细胞的染色体2n=48,温度适应范围4°～38℃,最适生长温度27℃,分裂指数在36小时时达最高值。经病原病毒的人工侵染等试验表明,AHZC 88细胞不受草鱼出血病病毒感染。在电镜切片观察中,细胞内不见病毒颗粒。LDH同工酶分析,比敏感细胞少1条酶带。     

循环水养殖系统中养殖密度对松江鲈生长的影响

为研究配合饲料条件下循环水养殖系统(RAS)中养殖密度对松江鲈生长的影响,选取体长为(2.97±0.12)cm、体质量为(0.26±0.03)g的松江鲈,分别按40尾/m²(A组)、80尾/m²(B组)和120尾/m²(C组)共3个养殖密度,在RAS中进行了为期240 d的养殖试验。试验结果显示：A组鱼的终末体质量、终末体长、体质量日增长量、存活率等均显著高于其他两组,A组鱼的体长日增长量显著高于C组(P<0.05);不同密度组间鱼体肥满度无显著性差异(P>0.05)。试验组单位面积产量由高到低依次为：C组(2.83 kg/m²)、B组(2.51 kg/m²)、A组(1.72 kg/m²)。试验组鱼体质量与体长均呈幂函数相关(m=aL^b,a=0.007 6～0.008 9,b=3.123 6～3.209 4),体长、体质量生长均以三次函数拟合较好。各组间的鱼体长、体质量变异系数均差异显著(P<0.05),其中B组最小...     

松嫩平原碱水水域对虾体重生长速度与Ca~(2+)、Mg~(2+)的关系

以盐碱化湖泊和池塘水进行凡纳滨对虾(Litopenaeus vannamei Boone,1931)幼虾生长试验,探讨碱水环境对虾体重生长速度与Ca2 、Mg2 、Ca2 Mg2 质量浓度及(1/2Ca2 )/(1/2 Mg2 )的关系,为碱水水域养殖提供科学依据.结果表明,在碱度为10.32～38.50 mmol/L、盐度1.02～3.34g/L、pH 8.5～9.5的水环境中,体长为10～20 mm的幼虾饲养27 d的体重生长速度为(0.95 0.18)mg/d,存活率(47.5±14.5)%.不同水环境存活率差异显著(P＜0.01),体重生长速度差别不明显(P＞0.05).体重生长速度与环境因子问的相关性不明显.通过驯化提高幼虾对水环境的综合适应能力,是碱水水域养殖对虾的重要途径.     

Development and characterization of five novel cell lines from snubnose pompano,Trachinotus blochii (Lacepede, 1801), and their application in gene expression and virological studies

Five novel permanent cell lines have been established from gill, heart, kidney, eye and fin of snubnose pompano, Trachinotus blochii. They were designated as snubnose pompano gill (SPG), snubnose pompano heart (SPH), snubnose pompano kidney (SPK), snubnose pompano eye (SPE) and snubnose pompano fin (SPF), respectively. All these cell lines were characterized and cryopreserved successfully at different passage levels. Cell lines were passaged every alternate day; SPG, SPH, SPK, SPE and SPF cell lines attained passage levels of 68, 74, 82, 79 and 106, respectively, since the initiation of their development in 2019. The cell lines grew well in Leibovitz's 15 medium containing 15% foetal bovine serum at 28°C. Immunophenotyping of the cell lines revealed the presence of fibronectin and pancytokeratin. No mycoplasma contamination was found. The transfection study revealed the gene expression efficiency of these cell lines by expressing the green fluorescent protein (GFP). The authentication on origin of cell lines from T. blochii was confirmed by amplification of species-specific mitochondrial cytochrome oxidase I gene. The results showed the susceptibility of these cell lines to fish nodavirus (FNV) and tilapia lake virus (TiLV) and resistance to cyprinid herpesvirus 2 (CyHV-2). The FNV infection in the cell lines was confirmed by RT-PCR, Western blot, ELISA and immunocytochemistry, while TiLV infection was confirmed by RT-PCR assay. These results revealed that these cell lines are suitable for virological and foreign gene expression studies.     

基于病毒mRNA监测的传染性脾肾坏死病毒灭活快速检验方法建立及其应用

为建立传染性脾肾坏死病毒(ISKNV)灭活快速检验方法,从ISKNV感染CPB细胞系转录谱筛选并经q RT-PCR验证表达量最高的病毒ORF099基因作为快速检测靶基因。以质粒p MDORF099为标准品,采用q PCR方法绘制了CT值与质粒拷贝数的标准曲线,其线性方程为CT=–3.42lgx+39.455,最低检测限为3拷贝/μL,结果显示组间和组内变异系数均小于2%,表明该方法具有较高的灵敏度和较好的重复性。将ISKNV病毒悬液10倍稀释成100~103拷贝/m L,分别取1 m L病毒稀释液接种CPB细胞,在第7、9和11天提取细胞总RNA,经基因组DNA去除试剂盒去除残留DNA后采用qRT-PCR方法检测ORF099基因转录本,结果显示在第7天即可从接种1个拷贝病毒的细胞中检测出ISKNV ORF099转录本。将3个浓度梯度(0.05%、0.1%、0.2%)的甲醛灭活ISKNV制备的模拟样品以及实验室制备的3批次ISKNV细胞灭活疫苗样品接种CPB细胞9 d,采用上述快速检验方法进行检测。0.05%、0.1%甲醛灭活模拟样品可检测到ISKNV ORF099基因转录本,其他样品均未检测到。而细胞盲传实验显示,0.1%终浓度甲醛灭活ISKNV接种细胞盲传3代未出现CPE,鱼体安全实验显示接种鱼体后无临床发病症状和实验鱼死亡,表明本研究建立的病毒灭活快速检验方法比细胞盲传法和鱼体安全实验具有更高的灵敏度,与传统检测方法相比具有灵敏度高、耗时短、检测效率高等优点,对提高ISKNV灭活疫苗生产效率具有重要意义。     

Establishment of cell lines from a tropical grouper,Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses

Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.     

Comparative sensitivity of five fish cell lines to wild type infectious haematopoietic necrosis virus from two Oregon sources

Abstract. Five fish cell lines (CHSE-214, STE-137, RTG-2, EPC and FHM) were compared for sensitivity to infectious haematopoietic necrosis virus (IHNV) from samples obtained from naturally-infected fish. Infectious ovarian fluids were obtained from steelhead trout, Salmo gairdneri Richardson, at the Round Butte Hatchery in central Oregon and tissue homogenates were prepared from chinook salmon, Oncorhynchus tshawytscha (Walbaum), alevins during an IHN virus epizootic at the Elk River Hatchery in coastal Oregon. The only lines to show characteristic viral cytopathology by plaque or end-point dilution assay for the steelhead trout virus isolate were the EPC and FHM cell lines. The chinook salmon isolates produced CPE in CHSE-214, STE-137, FHM and EPC cells. The titre of the salmon virus isolate was 10-50-fold higher on FHM and EPC cells by both assay methods. Neither by end-point nor plaque assay did the Round Butte or Elk River isolates produce CPE on RTG-2 cells. With both virus isolants both cell lines showed that greater sensitivity was obtained with plaque assay than with end-point titration. Pre-treatment of the cells with the polycation, polybrene, did not increase the virus titre in either assay. However, a transient enhancement in virus titre was observed in polybrene-treated STE-137 and CHSE-214 cells.     

Two iridovirus-susceptible cell lines established from kidney and liver of grouper, Epinephelus awoara (Temminck & Schlegel), and partial characterization of grouper iridovirus

Two iridovirus-susceptible cell lines were established and characterized from grouper Epinephelus awoara kidney and liver tissues. These cell lines have been designated GK and GL, respectively. The cells multiplied well in Leibovitz's L-15 medium, supplemented with 10% foetal bovine serum, at temperatures between 20 and 32 °C, and have been subcultured more than 120 times, becoming continuous cell lines. The cell lines consist of a heterogeneous mixture of fibroblastic and epithelial cells. The viability of cells, stored frozen in liquid nitrogen (−196 °C), was 95% after 1 year. Chromosome morphologies of GK and GL cells were homogeneous. Both cell lines were susceptible to grouper iridovirus, and yielded high titres of up to 108 TCID50 mL⁻¹. In addition, both cell lines effectively replicated the virus, which could be purified to homogeneity by cesium chloride gradient centrifugation. Electron microscopy studies showed that purified virus particles were 170±10 nm in diameter, and were hexagonal in shape. Virus-infected cells showed an abundance of virus particles inside the cytoplasm. These results show that the GK and GL cell lines effectively replicate grouper iridovirus, and can be used as a tool for studying fish iridoviruses.     

暗纹东方鲀线粒体DNA控制区结构和系统发育分析

以暗纹东方(Takifugu fasciatus)肝脏线粒体DNA为模板,参照GenBank中红鳍东方(Takifugu rubripes)线粒体DNA序列设计合成特异引物进行PCR扩增,获得了暗纹东方线粒体DNA控制区基因(818 bp)及5′端上游的tR-NAPro基因(71 bp)的全序列。控制区碱基组成为T32.2%,C 19.1%,A35.6%,G13.2%。对照其他已报道的鱼类控制区结构,对暗纹东方控制区的结构进行了分析,识别了其终止序列区、中央保守区和保守序列区,找到了终止相关的序列TAS以及保守序列(CSB1,CSB2,CSB3)。CSB1、CSB2序列相对保守,TAS与其回文基序可形成稳定的茎环结构,成为H-链复制延伸时的终止识别位点。同时运用DNA分析软件对暗纹东方与GenBank中其他10多种鱼类的mtDNA控制区序列进行比对,并选取东方属的7种鱼类mtDNA控制区序列构建分子系统树。结果显示控制区基因较适于科鱼类中同属不同种的系统发育分析。     

三种水生动物细胞系对两株蛙病毒敏感性的比较

利用3个不同物种的水生动物细胞系,包括爪蟾肾细胞系(A6)、大鲵胸腺细胞系(GSTC)和鲤上皮瘤细胞系(EPC),分别用沼泽绿牛蛙蛙病毒(RGV)和大鲵蛙病毒(ADRV)感染,进一步研究细胞病变显微形态、病毒滴度、细胞病变与不同感染时间的相关性等。结果显示,在光镜下可见感染病毒的细胞发生病变,A6和EPC细胞肿胀或破裂;GSTC细胞收缩或聚在一起形成多层。同种水产动物细胞系对不同蛙病毒的敏感性不同,在A6、EPC和GSTC细胞中,RGV的滴度分别为10~(3.6)、10~(5.9)和10~(6.6) TCID_(50)/m L;ADRV的滴度分别为10~(4.3)、10~(5.4)和10~(6.1) TCID_(50)/m L,表明GSTC细胞系对两种蛙病毒都更敏感。研究为后续蛙病毒致病机理提供了有用的信息和重要实验材料。     

凡纳滨对虾群体自交与杂交子代幼体对低温、低盐抗逆性与生长比较

为提高凡纳滨对虾种苗质量,探索群体选择育种和杂交育种方法在凡纳滨对虾大规模人工育苗生产中的应用,对1个经过人工选择的凡纳滨对虾群体A的自交和杂交子代的抗逆性和生长性状进行了比较。群体A是2008年来自美国种虾的子一代,并经过2个世代的人工选择和自交传代。群体B为2010年来自广东的繁育群体。A群体自交,及其与B群体的正、反杂交,产生3组不同交配组合的子代。对3个组合后代仔虾幼体的抗低温、低盐能力及养成期的生长性状进行比较分析,结果表明,以A群体为母本的杂交组AB抗逆性最强,以B群体为母本的杂交组BA抗逆性次之,而A群体自交组抗逆性最弱。在低温(13.0±0.9) ℃和低盐4.7条件下,AB组存活率分别比AA组高27.6%和64.4%。3个组合养成期的体质量生长速度为AB组(253±55) mg/d、 BA组(208±52) mg/d、AA组(219±36) mg/d,AB组比BA组快21.6%,比AA组快15.5%。研究表明,凡纳滨对虾的杂交优势与亲本的提纯、选优密切相关。     

Infectivity study of Streptococcus phocae to seven fish and mammalian cell lines by confocal microscopy

Streptococcus phocae is a beta-haemolytic bacterium that causes systemic infections in Atlantic salmon, Salmo salar L., cultured in southern Chile and also in seals. In this study, the host-pathogen interaction between S. phocae and seven types of cell lines (fish and mammalian) was examined using an indirect fluorescent antibody and confocal microscopy (CM). Chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), salmon head kidney (SHK-1) and Atlantic salmon kidney were used as the fish cell lines, while human cervix epithelial adenocarcinoma (HeLa), African green monkey kidney fibroblast (Cos-7) and mouse leukaemic monocyte macrophage (Raw 264.7) were included as mammalian cell lines. Streptococcus phocae type strain ATCC 51973(T) and isolates LM-08-Sp and P23 were selected as representatives from the salmon and seal host, respectively. For the CM examination, monolayers seeded on round coverslips were studied at 2- and 20-h post-inoculation (pi). The results showed that there is no common infectivity pattern between the three S. phocae strains at 2-h pi and the cell lines tested, regardless of the source of isolation (seal or salmon). All S. phocae strains could internalize and were found inside the fish and mammalian cell cytoplasm after 20-h pi. Regardless of the cells studied (fish or mammal) and incubation (2 and 20 h), S. phocae was never observed inside the nuclei. Seal and salmon isolates showed the highest number of bacteria entering into the primate cell lines (HeLa and Cos-7) from 2-h pi, while ATCC 51973(T) was not found outside or inside the HeLa and Cos-7 cells.