绿色荧光蛋白基因重组子的构建及其在鱼类受精卵中的表达

通过体外重组，将绿色荧光蛋白基因编码序列克隆到鲤鱼肌动蛋白基因启动子下游，构建成能在鱼体内表达绿色荧光蛋白的重组分子。用限制性内切酶PvuI酶切线性化后，通过显微注射法导入金鱼受精卵内，在转化后48h，可观察到绿色荧光，经PCR初步筛选后，对显阳性个体进行Southern杂交检测，结果表明，转化个体表现出较强的杂交信号。这说明绿色荧光蛋白基因能在金鱼体内整合、表达。     

Characterization and viral susceptibility of a brain cell line from brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål) with persistent betanodavirus infection

A continuous cell line designated BMGB (brown‐marbled grouper brain) was established from the brain tissues of the brown‐marbled grouper Epinephelus fuscoguttatus and characterized. BMGB cells were identified as astroglial progenitor cells because they expressed glial fibrillary acidic protein and keratin and were persistently infected by betanodavirus, as confirmed through immunocytochemistry, polymerase chain reaction and immunoblot analyses. Because few intact virions were present in the BMGB cell culture fluid, the cytopathic effect (CPE) was not observed when the culture fluid was inoculated with GBC1 cells. However, BMGB cells displayed typical CPE after infection with additional betanodavirus, megalocytivirus and chum salmon reovirus. BMGB cells showed low myxovirus resistance (Mx) protein expression, which increased following betanodavirus and reovirus infection. Because the cells contained several unusual or degraded viral proteins, the persistent infection of betanodavirus in the BMGB cells may have resulted from a mechanism that destroys the viral proteins rather than the result of Mx protein expression. Despite the persistent betanodavirus infection, BMGB cells proliferated in a manner similar to other normal tropic fish cells and supported the propagation of several piscine viruses; however, the yield was lower than that of normal cells. The BMGB cells will be useful for investigating virus and host cell interaction.     

用绿色荧光蛋白标记的细菌研究鱼体吸收颗粒抗原的部位

用绿色荧光蛋白标记的嗜水气单胞菌４３３２株（Ａｈ４３３２＾ＧＦＰ）菌液高渗浸泡和直接浸泡异育银鲫,研究鱼体对颗粒抗原的吸收。经两种方法处理的鱼,在整个实验过程中显示,鱼鳃的标记菌检出量均高于皮肤和肠道中的标记菌,证实鳃是抗原吸收的主要部位。另外,鱼体经高渗处理,鳃和皮肤可检出的标记菌明显增加。在肠道中,除浸泡６０ｍｉｎ,浸泡１０ｍｉｎ、３０ｍｉｎ及１２０ｍｉｎ的细菌检出量也明显增加。显示高渗可促进抗原的吸收。     

Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line

In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 degrees C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 degrees C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 degrees C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 degrees C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 degrees C) compared with the non-permissive temperature of 28 degrees C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis.     

Viral susceptibility,transfection and growth of SPB – a fish neural progenitor cell line from the brain of snubnose pompano,Trachinotus blochii (Lacépède)

This study investigates the susceptibilities of the SPB cell line to fish viruses including giant seaperch iridovirus (GSIV‐K1), red sea bream iridovirus (RSIV‐Ku), grouper nervous necrosis virus (GNNV‐K1), chum salmon reovirus (CSV) and eel herpesvirus (HVA). GSIV‐K1, RSIV‐Ku and CSV replicated well in SPB cells, with a significant cytopathic effect and virus production. However, the cells were HVA and GNNV refractory. To examine the ability of SPB cells to stably express foreign protein, expression vectors encoding GNNV B1 and B2 fused to enhanced green fluorescent protein (EGFP) and GSIV ORF35L fused to DsRed were constructed and introduced by transfection into SPB cells. Stable transfectants displayed different morphologies compared with SPB and with each other. EGFP‐B1 was predominantly localized in the nuclei, EFPF‐B2 was distributed throughout the cytoplasm and nucleus, and granular 35L‐DsRed was localized with secreted vesicles. The expression of EFPF‐B2 in SPB cells produced blebs on the surface, but the cells showing stable expression of EGFP, EGFP‐B1 or 35L‐DsRed showed normal morphologies. Results show the SPB cells and the transfected cells grow well at temperatures between 20 and 35 °C and with serum‐dependent growth. SPB cells are suitable for studies on foreign protein expression and virology.     

The use of green fluorescent protein as a marker for monitoring a probiotic Bacillus S11 in the black tiger shrimp Penaeus monodon

To monitor the probiotic Bacillus S11 (BS11) in vivo , wild-type cells were transformed with the green fluorescent protein (GFP)-expressing plasmid, pAD44-12, carrying the gfp mut3a gene under the constitutive Bacillus cereus UW85 promoter, and its non-GFP control plasmid pAD. Transformants with pAD44-12 (BS11-GFP), not pAD (BS11-pAD), expressed detectable but not too high levels of green fluorescence. Chloramphenicol resistance (BS11-GFP, BS11-pAD) and GFP fluorescence (BS11-GFP) as markers suggested that plasmid retention was 78–79% for both BS11-GFP and BS11-pAD cells after approximately 50 generations of growth without antibiotic selection. When mixed into shrimp feed at a final concentration ∼10⁵ CFU g⁻¹, the inclusion of viable transformed bacteria in fed shrimps was observed. After feeding shrimp three times daily in 400-L cement tanks for 9 weeks, no significant differences in the average shrimp weight, the number of BS11 in either the culture water or in the shrimp's gut were seen between shrimp fed BS11-GFP and BS11-pAD or BS11, suggesting that expression of the gfp mut3a gene has no detectable effect on BS11 properties and shrimp growth. Histological examination of sections of shrimp's intestines following feeding with BS11-GFP demonstrated that BS11-GFP in shrimp feed survived and adhered onto the shrimp intestines' surface. BS11-GFP thus has good potential as a non-invasive marker tag for short-term experiments.     

穿膜肽引导的体外表达转录因子蛋白Sox2进入红鳍东方鲀精巢细胞系

为了探索适用于体外培养的鱼细胞外源基因转入方法, 本研究通过构建红鳍东方鲀(Takifugu rubripes)转录因子Sox2的重组表达载体pET32a(+)-Sox2-11R-6His, 诱导表达并纯化得到了C末端连接多聚精氨酸(11R)的重组蛋白Sox2-11R-6His, 以其与红鳍东方鲀精巢细胞系细胞共孵育12 h后, 光镜观察结合Western Blot检测发现重组蛋白进入细胞的效率与浓度呈剂量依赖关系且最佳孵育浓度为8 μg/mL, 当重组蛋白质量浓度达到10 μg/mL时, 表现出明显的细胞毒性。对外源蛋白进行免疫荧光标记定位, 发现重组蛋白分布于细胞质, 部分进入到细胞核中。证明了穿膜肽11R可以有效运载转录因子重组蛋白至红鳍东方鲀的细胞系细胞中。本研究旨在将广泛应用于哺乳动物的细胞基因递送载体穿膜肽应用于鱼类细胞系细胞。

     

Propagation of yellow grouper nervous necrosis virus (YGNNV) in a new nodavirus-susceptible cell line from yellow grouper, Epinephelus awoara (Temminck & Schlegel), brain tissue

A nodavirus was isolated from diseased yellow grouper, Epinephelus awoara , larvae cultured in southern Taiwan. The histopathology and RT–PCR results confirmed that it was a fish nodavirus; its coat protein gene sequence was similar to that of red spotted grouper nervous necrosis virus (RGNNV) and it is named yellow grouper nervous necrosis virus (YGNNV). A new nodavirus-susceptible cell line, grouper brain (GB) was established and characterized from the brain tissue of yellow grouper. The GB cells multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum at temperatures between 24 and 32 °C, and have been subcultured more than 80 times, becoming a continuous cell line. The GB cell line consists of fibroblast-like cells and some epithelioid cells. The cell line yielded titres of YGNNV up to 10^8.5 TCID₅₀ mL^–1. The GB cells effectively replicated the virus at 28 °C, which could be purified to homogeneity by caesium chloride gradient centrifugation. Electron microscopy studies showed that purified virus particles were 25–30 nm in diameter. The cytoplasm of infected cells was filled with aggregates of virus particles. These results indicate that the GB cell line is a significant tool for the study of fish nodaviruses.     

罗非鱼肾脏细胞系的建立及其生物学特性

采用组织块移植法,对尼罗罗非鱼（Oreochromis niloticus）的肾脏组织细胞进行原代培养,建立了罗非鱼肾脏细胞系,已稳定传代培养50代以上,命名为TiK。罗非鱼肾脏细胞系为纤维样细胞,其最佳培养基为DMEM,最适培养温度为28℃,最适血清浓度为15%。在最适培养条件下,罗非鱼肾脏细胞系的群体倍增时间为45.8 h。细胞经液氮冷冻保存6个月后进行复苏,经台盼蓝染色,约（89.84±3.48）%的细胞具有活性,复苏后细胞生长旺盛。染色体分析显示,第32代罗非鱼肾脏细胞系染色体数目分布在20~66之间,众数为48。使用本实验室分离鉴定的罗非鱼病毒感染罗非鱼肾脏细胞,可产生典型的细胞病变效应,表明罗非鱼肾脏细胞对该病毒敏感。该细胞系的建立为罗非鱼病毒病防控技术研究提供了重要的实验材料。     

Establishment and characterization of a fibroblast cell line derived from the dorsal fin of red sea bream,Pagrus major (Temminck & Schlegel)

The establishment and partial characterization of a continuous cell line from the dorsal fin of red sea bream, Pagrus major, are described. The cell line, designated RSBF‐2, has been subcultured for more than 100 passages since its initiation in November 2000. It was optimally maintained at 28 °C in Leibovitz L‐15 medium with 10% foetal bovine serum. Propagation of RSBF‐2 cells was serum dependent and exhibited low plating efficiency (<1.7%). Aside from long‐term cryopreservation, the cells could also be kept at 4 °C for 72 days. The distribution of the chromosome number was 38–98 with a mode of 48. The RSBF‐2 cell line was susceptible to red sea bream iridovirus but only produced a few rounded and refractory cells. Virus‐inoculated RSBF‐2 cells were then subcultured to generate a persistently infected cell line. RSBF‐2 was also very sensitive to the extracellular products of Photobacterium damselae ssp. piscicida and produced significant fluorescent signals after transfection with pEGFP‐C3. Analysis of mitochondrial cytochrome b gene sequences revealed 99% identity between the cell line and Pagrus major.     

Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

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.     

剑尾鱼脑细胞系的建立及细胞色素P4501A的诱导表达

为了构建剑尾鱼脑细胞系并探讨其细胞色素P4501 a(CYP1A)基因的诱导效应,实验通过胰蛋白酶消化法对剑尾鱼脑组织进行体外培养,经连续继代培养,建立了可稳定传代的脑细胞系,命名为SFB.SFB最适培养液为含有15％胎牛血清(FBS)的DMEM/F-12和L-15等比混合培养液,培养条件为27℃,5％ CO2.生长特性研究表明,第65代细胞的群体倍增时间为43.048h,显示出旺盛的生长和分裂能力.染色体分析发现,培养细胞的染色体众数为48条,SFB核型公式为2n =2st +46t,臂指数(NF) =48,与剑尾鱼一致.诱导实验表明,SFB在10-8～ 10-5 mol/L的苯并(a)芘诱导下,CYP1A mRNA表达量显著提升,且表现出良好的剂量效应关系.脑细胞系的建立为剑尾鱼的毒理学评价研究提供了便利,也为其系统的生态毒理学应用打下基础.     

Development of a continuous cell line from larval Atlantic cod (Gadus morhua) and its use in the study of the microsporidian,Loma morhua

In vitro cell culture methods are crucial for the isolation, purification and mass propagation of intracellular pathogens of aquatic organisms. Cell culture infection models can yield insights into infection mechanisms, aid in developing methods for disease mitigation and prevention, and inform commercial‐scale cultivation approaches. This study details the establishment of a larval cell line (GML‐5) from the Atlantic cod (Gadus morhua) and its use in the study of microsporidia. GML‐5 has survived over 100 passages in 8 years of culture. The line remains active and viable between 8 and 21°C in Leibovitz‐15 (L‐15) media with 10% foetal bovine serum and exhibits a myofibroblast phenotype as indicated by immuno‐positive results for vimentin, α‐smooth muscle actin, collagen I and S‐100 proteins, while being desmin‐negative. GML‐5 supports the infection and development of two microsporidian parasites, an opportunistic generalist (Anncaliia algerae) and cod‐specific Loma morhua. Using GML‐5, spore germination and proliferation of L. morhua was found to require exposure to basic pH and cool incubation temperatures (8°C), in contrast to A. algerae, which required no cultural modifications. Loma morhua‐associated xenoma‐like structures were observed 2 weeks postexposure. This in vitro infection model may serve as a valuable tool for cod parasitology and aquaculture research.     

Development of a black sea bream fibroblast cell line and its potential use as an <Emphasis Type="Italic">in vitro</Emphasis> model for stress protein studies

A fibroblast cell line (BSF) derived from a caudal fin explant of black sea bream (Mylio macrocephalus) was developed. The optimum fetal bovine serum (FBS) concentration for fibroblast cell line growth was found to be 15–20% v/v FBS and the optimum temperature range for growth was found to be 26–30 °C. The fibroblast cells displayed a diverse distribution in chromosome number with two modal chromosome numbers of 48 and 54. Upon acute heat shock (+8 °C) the cells displayed a 4.1 fold increase in hsp70 and this elevation was not prolonged as hsp70 returned to near basal levels following a 6 h recovery period. The effect of the hsp70 inducer L-azetidine- 2-carboxylic acid was tested and it was found that at a concentration of 10 mM this inducer caused a 2.3 fold increase in hsp70 levels. The sensitivity of the fibroblast cell line to heavy metal exposure was tested by treatment with Cu2+ and it was found that hsp70 was significantly elevated in the presence of micromolar concentrations of Cu2+. The data from this study demonstrates that the established black sea bream fibroblast cell line could serve as a useful in vitro model for stress protein studies.     

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.