title: Molecular structure, expression and bioactivity characterization of TNF13B (BAFF) gene in mefugu, Takifugu obscurus.
abstract: B-cell activating factor of the TNF family (BAFF) induces B-cell survival, proliferation, immunoglobulin secretion and plays a role in enhancing immune responses. In the present study, a BAFF homolog has been identified in mefugu Takifugu obscurus, and its biological activities have been characterized. The mefugu BAFF (fBAFF) cDNA is 789 bp in length and translates into a 262-aa protein. The fBAFF genomic sequence consists of six exons and five introns, is approximately 1.8 kb in size. Amino acid sequence comparison indicated that fBAFF possessed the TNF signatures, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues, which were the typical characteristics of TNF gene in mammals and birds. The predicted three-dimensional (3D) structure of the fsBAFF monomer analyzed by comparative protein modeling revealed that it was very similar to its human counterpart. Real-time quantitative PCR (qPCR) analysis revealed that fBAFF was predominantly expressed in mefugu lymphoid tissue spleen. The SUMO-fsBAFF and GFP/fsBAFF efficiently expressed in Escherichia coli Rosetta (DE3) were confirmed by SDS-PAGE and Western blotting analysis. After purification, the GFP/fsBAFF fusion protein obtained similar fluorescence spectrum to those of GFP. Laser scanning confocal microscopy analysis showed GFP/fsBAFF could bind to its receptors. In vitro, the MTT assays and flow cytometric analysis indicated that SUMO-fsBAFF could promote the proliferation of mefugu splenocytes or mouse splenic B cells together with/without anti-mouse IgM. These findings indicate that SUMO-fsBAFF plays an important role in proliferation of mefugu B cells and has functional cross-reactivity among mefugu and other mammalians. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in fish.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Hongxin
authors.initials: H
authors.lastname: Ai
authors.forename: Yuefen
authors.initials: Y
authors.lastname: Shen
authors.forename: Cui
authors.initials: C
authors.lastname: Min
authors.forename: Shuying
authors.initials: S
authors.lastname: Pang
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Zhang
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Zhao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053264:B-Cell Activating Factor / Q000235:genetics* / Q000378:metabolism*; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D016000:Cluster Analysis; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005434:Flow Cytometry; D040461:Gene Components; D049452:Green Fluorescent Proteins; D051379:Mice; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D013154:Spleen / Q000378:metabolism; D023741:Takifugu / Q000235:genetics*; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: D053264:B-Cell Activating Factor; D017931:DNA Primers; D018076:DNA, Complementary; D013778:Tetrazolium Salts; D013844:Thiazoles; D049452:Green Fluorescent Proteins; C022616:thiazolyl blue