| Aquaculture, which has great prospects and economic value, has developed rapidly in recent years. As an valuable aquaculture species, turbot farming area are expanded continuously. However, infectious disease, espically viral diseases with high mortalities of turbot occurred because of increasing water pollution and the intensive aquaculture. Mass mortality of turbot brings enormous economic losses. Viral disease usually outbreak rapidly, with high mortality and are difficult to control. The establishment of turbot cell line is a good method for isolation, identification and characterization of infectious viruses. It is very useful for epidemiology of viral infections and characterization of the viruses in turbot. But now there are only several turbot cell lines in the world which are kept in several Labs. This is really far not enough for turbot virology, toxicology, immunology, physiology, genitics and germplasm conservation. This is very bad for treatment of turbot virus disease. So there is urgent need to establish turbot cell line for Lab research.In this experiment, the fin tissue was digested with hyaluronidase (2mg/ml) and typeⅡcallagenase (2mg/ml). By culturing the cells in different conditions, it was concluded that the optimal medium, pH value and temperature for fin cell growth were Leibovitz-15 medium, pH 7.2 and 20-24℃. The turbot fin cells were conducted at pH 7.2 and 24℃using Leibovitz-15 medium which supplemented with 20% FBS, 10ng/ml basic fibroblast growth factor (bFGF) and 10ng/ml epidermal growth factors (EGF). The emergence of new cell growth was observed 2 weeks after the tissue explanting. The cells appeared uniformly fibroblast and grew fast. Twenty days later the cells grew into monolayer in 25 cm2 tissue culture flasks.The cell monolayer needed to be subcultured with trypsin-digestion method. The cells grew into monolayer in 3-5 days after they were subcultured in a 1/2 ratio. The cells at 25 passages has been cryopreserved in liquid nitrogen and can be recovered with very good cell viability. There is no difference between and after cryopreserved. The turbot fin cells at 30 passage were at stagnation stage in the first 1 day after subculture and at logarithmic stage in 2-4 days and 5-6 days is the stable stage. The doubling time of turbot fin cells is 50.7 h. The chromosome number of turbot fin cells was varied from 26-58 and the 75.97% turbot fin cells had a chromosome munber is 44.The major envelope protein VP28 was extracted from WSSV gene and inserted into the PEGFP-N1 vector. The pEGFP-N1-VP28 vector was transfected into the turbot fin cells using LipofectamineTM2000. The transfected cells were observed in fluorescence microscope after 24 hours. There was fluorescence in these turbot fin cells and the pEGFP-N1-VP28 was expressed. This turbot fin cell line can be used for eukaryotic expression of viral proteins.The primary culture of tissues of turbot, verasper variegates, commom sole and Penaeus chinensis was carried out. The tissues included:spleen, kidney, heart and intestinal of turbot; fin, heart,spleen of verasper variegates; heart, kidney of commom sole and blood and hepatopancreas cells of penaeus chinensis. Fin tissue was digested with hyaluronidase (2mg/ml) and typeⅡcallagenase (2mg/ml). Hepatopancreas cells of penaeus chinensis was digested with trypsin (0.25%). Other tissues were primary cultured with explanting. The cells from all tissues grew from the edges of seeded tissue explants in 2 weeks. The cells from turbot liver tissue explants and verasper variegates spleen tissue explants were subcultured using trypsin. The cells from verasper variegates spleen tissue explants were subcultured to 6 passages. The blood cells from penaeus chinensis survived over 1 week and the hepatopancreas cells survived for 2 days. |
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