Red sea bream brain cell line and its construction method and application
A technology of brain cells and red sea bream, applied in the biological field, can solve the problems of vacuolation of the brain and retina
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Embodiment 1
[0053] The construction of embodiment 1 red sea bream brain cell line
[0054] 1. Treatment of brain tissue: First, a healthy red sea bream with a body length of about 8 cm (purchased from a farm in Yangjiang City, Guangdong Province) was soaked and disinfected with 75% alcohol for 2 minutes. Dissect and remove the brain tissue in the workbench, roughly cut the brain tissue into 50-100 mm3 tissue blocks in a small glass bottle filled with HBSS (Hank's Balanced Salt Solution), collect the tissue blocks by centrifugation at 600 rpm, and repeatedly Centrifuge 3 times.
[0055] 2. Primary culture: put the tissue block into serum-containing collagenase digestion solution (digestion solution contains 10v / v% fetal bovine serum, 0.1w / v% type Ⅰ collagenase, 200U / mL penicillin, 200μg / mL mL streptomycin, 0.5 μg / mL amphotericin B in L-15 culture medium) for 6 hours at 28°C, after digestion, centrifuge at 1200 rpm for 5 minutes, collect the digested tissue cell suspension, Add 5 millilit...
Embodiment 2
[0059] The characteristics of embodiment 2 red sea bream brain cells
[0060] 1. Cell cryopreservation and recovery
[0061] Cell cryopreservation: From the 5th passage to the 65th passage, the cells are frozen every 5th passage. When the subcultured red sea bream brain cells grew to the logarithmic growth phase, they were stored in a liquid nitrogen tank for long-term storage in a -80°C refrigerator overnight using a programmed cooling box.
[0062] Cell recovery: Pick frozen cells at passages 30, 40, 50, and 60, take them out of the liquid nitrogen tank after 6 months, 1 year, and 2 years, and place them in a 37°C water bath to thaw quickly. Transfer the cell suspension into a cell culture flask, add 5 mL of M199 medium containing 10% FBS, culture overnight at 28°C, and replace with fresh medium the next day. After the cells were thawed, a small amount of cell suspension was taken in a centrifuge tube, stained with trypan blue, observed under a microscope, and the cell sur...
Embodiment 3
[0072] Example 3 Nervous necrosis virus (Nervous necrosis virus, NNV), grouper iridovirus (Singapore grouper iridovirus, SGIV), infectious spleen kidney necrosis virus (infectious spleenkidneynecrosis virus, ISKNV), mandarin fish bullet virus (Siniperca chuatsirhabdovirus, SCRV) ), Tilapia Lake Virus (TiLV) infection sensitivity of red sea bream brain cells (BB cells)
[0073] The 55th passage BB cells in the logarithmic growth phase were taken. NNV was inoculated at 0.1MOI, SGIV, SCRV and TiLV were all inoculated at 0.01MOI, and incubated at 28°C for 1h; ISKNV was inoculated at 1MOI, incubated at 28°C for 2h, and then supplemented with 2 % serum M199, continue to culture at 28°C. The cytopathic condition was observed every day. When the CPE reached 80%, the virus liquid was harvested by repeated freezing and thawing twice at -80°C.
[0074] 1. Susceptibility to different viruses
[0075] (1) Sensitivity of BB cells to NNV
[0076] The 55th passage BB cells in the logarithm...
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