title: 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 His6 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.
authors.affiliation: Key Laboratory of Ecology and Environmental Science of Guangdong Higher Education Institutes, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, School of Life Science, South China Normal University, Guangzhou, 510631, People's Republic of China, chengchanghong09@163.com.
authors.forename: Chang-Hong
authors.initials: CH
authors.lastname: Cheng
authors.forename: Fang-Fang
authors.initials: FF
authors.lastname: Yang
authors.forename: Shao-An
authors.initials: SA
authors.lastname: Liao
authors.forename: Yu-Tao
authors.initials: YT
authors.lastname: Miao
authors.forename: Chao-Xia
authors.initials: CX
authors.lastname: Ye
authors.forename: An-Li
authors.initials: AL
authors.lastname: Wang
authors.forename: Jin-Chang
authors.initials: JC
authors.lastname: Liu
authors.forename: Li-Wei
authors.initials: LW
authors.lastname: Liu
mesh_terms: D016980:Aeromonas hydrophila; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D005393:Fish Diseases / Q000378:metabolism; D005880:Gills / Q000378:metabolism; D016905:Gram-Negative Bacterial Infections / Q000378:metabolism / Q000662:veterinary; D060226:Head Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D011487:Protein Conformation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013154:Spleen / Q000378:metabolism; D023741:Takifugu
chemical_list: D018076:DNA, Complementary; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger