title: Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus.
abstract: LIGHT/TNFSF14 is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. In this study, the cDNA of mefugu (Takifugu obscures) LIGHT (designated as fLIGHT) was amplified from spleen by reverse transcription polymerase chain reaction. The open reading frame of fLIGHT covers 765 bp and translates into a 254-aa protein. The predicted three-dimensional (3D) structure of the soluble LIGHT of mefugu (designated as fsLIGHT) monomer analyzed by comparative protein modeling revealed that it was very similar to its human counterpart. Real-time quantitative PCR analysis indicated that LIGHT is constitutively expressed in various tissues in mefugu. The soluble LIGHT binding of green fluorescent protein (GFP) (designated as GFP/fsLIGHT) had been cloned into the pET28a vector; SDS-PAGE and western blotting analysis confirmed that the recombinant protein pET28a-GFP/fsLIGHT was efficiently expressed in Escherichia coli BL21 (DE3). After purification, the GFP/fsLIGHT fusion protein obtained similar fluorescence spectrum to those of GFP. Laser scanning confocal microscopy analysis showed GFP/fsLIGHT could bind to its receptors. In view of our basic research, LIGHT may be a potential immunologic factor for enhancing immunological efficacy in fish, and our results might provide a platform for further study into the effects of LIGHT.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, China.
authors.forename: Chunlan
authors.initials: C
authors.lastname: Li
authors.forename: Yuefen
authors.initials: Y
authors.lastname: Shen
authors.forename: DingFang
authors.initials: D
authors.lastname: Liang
authors.forename: Fei
authors.initials: F
authors.lastname: Yin
authors.forename: Hongxin
authors.initials: H
authors.lastname: Ai
authors.forename: Boqing
authors.initials: B
authors.lastname: Sun
authors.forename: Shikang
authors.initials: S
authors.lastname: Lin
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli; D029941:Fish Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D023741:Takifugu / Q000235:genetics*; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
chemical_list: D029941:Fish Proteins; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14
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