Abstract:Fish cells play an important role in virus isolation, identification, functional gene analysis, and biological product preparation. Mandarin fish Siniperca chuatsi is one of the most popular aquaculture species. With a rapid increase in production, the occurrence of diseases has also increased. Moreover, there are very few cell lines of mandarin fish that can be used for virus isolation and gene function identification. In this study, the body surface of mandarin fish was disinfected with povidone-iodine and 75% alcohol. The brain tissue was removed and washed in PBS buffer containing 2´antibiotic-antimycotic 4~5 times in a biosafety cabinet. The tissue was cut into blocks of approximately 3 mm3 with sterilized ophthalmic scissors in a sterile petri dish. After digestion at 37℃ for 0.5~1 h, the tissue blocks were cleaned with L-15 medium containing 10% fetal bovine serum (FBS). The suspension was centrifuged at 1000 r/min for 5 min, and the tissue blocks were transferred into a cell culture flask. A medium containing 30% FBS, penicillin, streptomycin, bFGF, and IGF was added to the cell culture flask, which was placed in an incubator at 28℃ for primary culture to establish the brain tissue cell line. To determine the growth properties of the new mandarin fish brain (MFB) cells, the cells were seeded onto 24-well plates with L-15 medium containing 10% FBS at an initial density of 5×104 cells per well. Cell growth rates were compared under different conditions, including five different media, four different serum concentrations, and four different temperatures. The optimal culture conditions for MFB cells were L-15 containing 10% FBS at 28℃. Under the optimal culture conditions, the doubling time of MFB cell number was ~46.6 h. Chromosome numbers for the 25th generation MFB ranged from 20 to 60, with a mode of 56 and frequency of 20%. Using the total genomic DNA of MFB cells as a template, specific primers were designed for the 28S rRNA gene. A partial gene fragment of 528 bp was obtained by PCR amplification, consistent with the expected size. The PCR-amplified fragments were sequenced and compared with the GenBank database by BLAST analysis. The sequence was consistent with that published in GenBank for the mandarin fish 28S rRNA gene (EF120974). Confirming that the cell line was derived from mandarin fish. NeuN (neuronal nucleus) is a neuro-specific nuclear regulatory molecule and a unique neuronal binding protein that is widely used in the study and diagnosis of neuronal antigens after mitosis. β-tubulin Ⅲ is a signature skeletal protein of mature neurons and is mainly distributed in the synapses and cytoplasm. The purity of MFB cells was determined by immunofluorescence cytochemistry (using β-tubulin and Neu-N). The results showed that all cultured MFB cells were neuron-like. Virus research requires the establishment of a sensitive cell line that allows for the proliferation of viruses in living cells. In this study, MFB cells were used to culture common aquatic viruses. The results of the virus sensitivity test showed that MFRaIV, LMBRaIV, and GSIV could infect and produce typical cytopathic effects in MFB cells, with titers of 108.68±0.12, 108.36±0.15, and 1010.15±1.85 TCID50/mL, respectively; thus, MFB cells are sensitive to MFRaIV, LMBRaIV, and GSIV. The replication level of GSIV in MFB cells was higher than that in MFRaIV and LMBRaIV. MFB cells were insensitive to ISKNV, GCrV-I, KHV, CEV, and CyHV-2. Transfection of exogenous genes into cells is a crucial step in gene function research. After 50 passages, Lipofectamine®2000 was used to transfer pEGFP-N1 into MFB cells. Green cells were observed under a fluorescence inverted microscope 48 h after transfection. The transfection efficiency was determined as the proportion of green positive cells in five random fields; the proportion of green cells was (22.20±1.72)%. In conclusion, a cell line derived from the brain tissue of mandarin fish was successfully established in this study. It is sensitive to a variety of aquatic animal viruses and can be used for gene transfection. It not only enriches the available resources of mandarin fish cells but also provides important experimental materials for further research into infection mechanisms and the development of virus and disease prevention technologies.
