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Int Immunopharmacol. 2013 Mar;15(3):478-87. doi: 10.1016/j.intimp.2013.02.006. Epub 2013 Feb 19.

Characterization of the molecular structure, expression and bioactivity of the TNFSF13B (BAFF) gene of the South African clawed frog, Xenopus laevis.

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1
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.

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.

PMID:
23428910
DOI:
10.1016/j.intimp.2013.02.006
[Indexed for MEDLINE]

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