Identification, characterization and functional analysis of anti-apoptotic protein BCL-2-like gene from pufferfish, Takifugu obscurus, responding to bacterial challenge

Abstract

Apoptosis plays a crucial role in many biological processes, including development, cellular homeostasis and immune responses. The BCL-2 family is a key regulator of the mitochondrial response to apoptotic signals in the intrinsic pathway. In this study, we identified and characterized the cDNA and expression pattern of pufferfish BCL-2 (PfBCL-2). The full-length cDNA of PfBCL-2 was 1412 bp with an open reading frame of 657 bp encoding a putative protein of 219 amino acids (Accession no: KP898414). The calculated molecular mass of the PfBCL-2 was 24.2 kDa with a predicted isoelectric point of 5.27. The deduced PfBCL-2 protein exhibited four highly conserved BCL-2 homology domains, suggesting that PfBCL-2 may play a similar role in the apoptotic-signaling pathway as in other species. Real-time PCR results showed that PfBCL-2 transcript was expressed in a wide range of tissues but exhibited the greatest level of expression in blood. Transcriptional responses of PfBCL-2 exhibited different spatial and temporal expression profiles in liver and blood after bacterial infection. PfBcl-2 transcript was significantly up-regulated in liver at 6, 12, 24 and 48 h (with maximum induction at 48 h) and was up-regulated in blood at 3, 6, 12 and 24 h (with maximum induction at 12 h). Meanwhile, recombinant PfBCL-2 fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified using Ni-nitrilotriacetic acid resin. Western blot analysis indicated that its protein level appeared to be elevated during the initial bacterial infection. These results suggest that PfBCL-2 plays important roles in immune responses against bacteria challenge