Cyprinid herpesvirus II-sensitive brain tissue cell line of Carassius auratus gibelio and establishing method and use thereof

A method for establishing a cyprinid herpesvirus II-sensitive brain tissue cell line, named GiCB, derived from a brain tissue of Carassius auratus gibelio, deposited in China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO:

• C2013179, comprising the steps of:
• (1) treatment of brain tissue:
• removing brain tissue of Carassius auratus gibelio under sterile conditions, and subjecting the brain tissue to sterile treatment to obtain tissue blocks of 50-100 mm3;
• (2) primary culture:
• digesting the tissue blocks of step (1) above with a trypsin solution formulated for tissue separation for 10-15 min, shaking the combined tissue blocks and trypsin solution for 3 to 4 times, adding an equal volume of a culture solution formulated for brain tissue cells of Carassius auratus gibelio, pipetting a resulting mixture uniformly, centrifuging and collecting resulting digested cells, removing supernatant, adding culture solution, pipetting resulting cell pellets, culturing an obtained cell suspension in a culture flask, and changing half an amount of the culture solution present every two days;
• (3) subculture:
• after the primary cultured brain tissue of Carassius auratus gibelio has grown into a monolayer, adding 0.25% W/V trypsin solution and standing a resulting mixture for 2 min, suspending the cells in a culture solution, subculturing the cells by inoculating the cells first in one flask, then dividing the cells into two flasks;
• after the cells form a cell monolayer again, subjecting the cells to a next passage culture according to the above subculture of the preceding portion of step (3), to obtain the cyprinid herpesvirus II-sensitive brain tissue cell line of Carassius auratus gibelio, GiCB;
• wherein said trypsin solution formulated for tissue isolation is a 0.5%-0.7% W/V trypsin solution;
• wherein cell culture and subculture is performed at a temperature of 25-28° C., and at a pH of 7.0-7.4;
• and
• wherein said culture solution formulated for brain tissue cells of Carassius auratus gibelio is an M199 culture medium containing 10-20% V/V fetal bovine serum, 10-20 ng/ml human basic fibroblast growth factor, 10-20 ng/ml human epidermal growth factor, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, pH 7.0-7.4.