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Enhanced viability of a nervous necrosis virus‐infected stable cell line over‐expressing a fusion product of the zfBcl‐x L and green fluorescent protein genes
Author(s) -
Chen SP,
Yang HL,
Lin HY,
Chen MC,
Wu JL,
Hong JR
Publication year - 2006
Publication title -
journal of fish diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 85
eISSN - 1365-2761
pISSN - 0140-7775
DOI - 10.1111/j.1365-2761.2006.00725.x
Subject(s) - biology , apoptosis , tunel assay , green fluorescent protein , programmed cell death , microbiology and biotechnology , viability assay , cell culture , fusion protein , western blot , grouper , necrosis , virus , virology , gene , biochemistry , genetics , recombinant dna , fish <actinopterygii> , fishery
Nervous necrosis virus (NNV) infection induces host cell apoptosis by an ill‐understood process. We utilized a fusion between enhanced green fluorescent protein (EGFP) and the zfBcl‐x L gene in GL‐av cells to select for zfBcl‐x L stable cell lines and to assess the effectiveness of the anti‐apoptotic protein Bcl‐x L in circumventing NNV‐induced cell death. Stable EGFP and EGFP‐Bcl‐x L ‐expressing clones were obtained at high purity within 2.5–3 months. In the latter, the EGFP‐Bcl‐x L fusion protein (approximately 58.2 kDa, as ascertained by Western blot) was predominantly targeted to mitochondria. We assayed for apoptosis in red‐spotted grouper NNV Tainan no. 1 (RGNNV TN1)‐infected cells with terminal deoxynucleotidyl transferase (TdT)‐mediated end labelling (TUNEL) of DNA at different virus doses. NNV infection of NNV Bcl‐x L GL‐av cell line revealed a protective effect, with a decrease in TUNEL‐positive cells of 7%, 8% and 31.8% at 24, 48 and 72 h, respectively. In addition, RGNNV infection of the Bcl‐x L GL‐av cell line revealed a protective effect, with an enhanced viability of 3%, 40% and 73% at 24, 48, and 72 h, respectively. We conclude that NNV‐induced apoptotic cell death can be lessened in transgenic grouper fish cells.