PUBLICATION
Molecular cloning, functional characterization and phylogenetic analysis of B-cell activating factor in zebrafish (Danio rerio)
- Authors
- Liang, Z., Kong, Y., Luo, C., Shen, Y., and Zhang, S.
- ID
- ZDB-PUB-100420-13
- Date
- 2010
- Source
- Fish & shellfish immunology 29(2): 233-240 (Journal)
- Registered Authors
- Keywords
- BAFF, cDNA cloning, Characterization, Evolution, Phylogeny, B-cell survival, 3D structure, Zebrafish (Danio rerio)
- MeSH Terms
-
- Amino Acid Sequence
- Animals
- B-Cell Activating Factor/chemistry
- B-Cell Activating Factor/genetics*
- B-Cell Activating Factor/immunology*
- Base Sequence
- Cells, Cultured
- Cloning, Molecular
- Gene Expression Profiling
- Gene Expression Regulation*
- Humans
- Mice
- Models, Molecular
- Molecular Sequence Data
- Phylogeny*
- Protein Structure, Tertiary
- Recombinant Proteins/immunology
- SUMO-1 Protein/immunology
- Sequence Alignment
- Zebrafish/classification*
- Zebrafish/genetics
- Zebrafish/immunology
- Zebrafish/physiology*
- PubMed
- 20382231 Full text @ Fish Shellfish Immunol.
Citation
Liang, Z., Kong, Y., Luo, C., Shen, Y., and Zhang, S. (2010) Molecular cloning, functional characterization and phylogenetic analysis of B-cell activating factor in zebrafish (Danio rerio). Fish & shellfish immunology. 29(2):233-240.
Abstract
B-cell activating factor (BAFF), belonging to the TNF family, is a critical cytokine for B-cell survival, proliferation, maturation and differentiation. In the present study we cloned the cDNA of zebrafish (Danio rerio) BAFF (designated zBAFF) by reverse transcription-PCR (RT-PCR). The open reading frame (ORF) of zBAFF consists of 807 bases encoding a protein of 268 amino acids. The deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, and three cysteine residues, which are the typical characteristics of TNF gene in mammals and birds. Phylogenetic analysis exhibits the highest identity score 67.6, 61.4 and 66.9% with the rainbow trout, tetraodon and salmon counterparts, respectively. The identity to avian and mammalian BAFFs ranges from 49.7 to 53.8%. Recombinant soluble zBAFF (zsBAFF) was fused with a small ubiquitin-related modifier gene (SUMO) to enhance the soluble expression level in Escherichia coli BL21 (DE3). The resulting fused protein SUMO-zsBAFF was highly expressed in BL21 (DE3) with a molecular weight of 38kDa. The fusing protein was purified using metal chellate affinity chromatography (Ni-NTA) and cleaved by a SUMO-specific protease, then confirmed by SDS-PAGE and Western blotting analysis. In vitro, the MTT assay indicated that the purified zsBAFF as well as SUMO-zsBAFF proteins were able to promote spleen lymphocyte survival in a dose-dependent manner also to co-stimulate the proliferation of mammalian B-cells with anti-IgM. Thus, the fusion protein represents a readily obtainable source of biologically active zsBAFF that may prove useful in further studies on zebrafish BAFF and its receptors.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping