title: Characterization of the molecular structure, expression and bioactivity of the TNFSF13B (BAFF) gene of the South African clawed frog, Xenopus laevis.
abstract: B cell activating factor (BAFF), a member of the tumor necrosis factor family, is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. In the present study, the full-length cDNA of BAFF from the South African clawed frog (Xenopus laevis, designated xlBAFF) was cloned using rapid amplification of cDNA ends (RACE) techniques and RT-PCR. The full-length cDNA of xlBAFF consists of 1204 bases including an open reading frame (ORF) of 801 nucleotides that are translated into a predicted 266 amino acid protein. Sequence comparison indicated that the amino acids of xlBAFF possessed the TNF signature, including a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the xlBAFF monomer revealed that it was very similar to its counterparts. Real-time quantitative PCR analysis revealed that xlBAFF could be detected in various tissues and predominantly expressed in the spleen and other lymphoid tissue. The soluble xlBAFF had been cloned into a pET28a vector to express the recombinant protein. The His6-xlBAFF was efficiently expressed in Escherichia coli. BL21 (DE3) and its expressions were confirmed by SDS-PAGE and Western blotting analysis. After purification, laser scanning confocal microscopy analysis showed that xlBAFF could bind to its receptors on B cells. CCK-8 assays revealed that xlBAFF is not only able to promote survival/proliferation of South African clawed frog lymphocytes but also able to stimulate survival/proliferation of mouse B cells. These results will allow for further investigation the use of X. laevis as an in vivo model for related studies.
authors: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors: Lin
authors: L
authors: Yang
authors: Lidan
authors: L
authors: Zhou
authors: Xicui
authors: X
authors: Zong
authors: Xiang
authors: X
authors: Cao
authors: Xuemei
authors: X
authors: Ji
authors: Wei
authors: W
authors: Gu
authors: Shuangquan
authors: S
authors: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053264:B-Cell Activating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism*; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D013154:Spleen / Q000166:cytology; D019076:Transgenes / Q000235:genetics; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis / Q000276:immunology*
chemical_list: D053264:B-Cell Activating Factor; C583528:TNFSF13B protein, Xenopus; D029867:Xenopus Proteins