Molecular cloning, expression and functional analysis of B-cell activating factor (BAFF) in yellow grouper, Epinephelus awoara

Mol Immunol. 2014 May;59(1):64-70. doi: 10.1016/j.molimm.2014.01.005. Epub 2014 Feb 1.

Abstract

B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation and immunoglobulin secretion. In this study, the yellow grouper (Epinephelus awoara) BAFF (designated EaBAFF) gene was cloned using RT-PCR and RACE (rapid amplification of cDNA ends) techniques. The full-length EaBAFF was 1442bp and contained an open reading frame of 780bp encoding a putative protein of 259 amino acids. Amino acids sequence comparison indicated that EaBAFF possessed the TNF signature. The soluble BAFF (EasBAFF) had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-EasBAFF was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, the WST-8 assay indicated that EasBAFF was not only able to promote the survival/proliferation of yellow grouper splenic lymphocytes but also able to promote the survival/proliferation of mouse splenic B cells. Our findings may provide valuable information for research into the immune system of E. awoara and EasBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in fish.

Keywords: B-cell survival; BAFF; RACE; Yellow grouper (E. awoara).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • B-Cell Activating Factor / classification
  • B-Cell Activating Factor / genetics*
  • B-Cell Activating Factor / metabolism
  • B-Lymphocytes / metabolism
  • Blotting, Western
  • Cloning, Molecular
  • Fish Proteins / genetics*
  • Fish Proteins / metabolism
  • Gene Expression*
  • Mice
  • Microscopy, Confocal
  • Molecular Sequence Data
  • Perciformes / genetics*
  • Perciformes / metabolism
  • Phylogeny
  • Protein Binding
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Spleen / cytology
  • Transcriptome

Substances

  • B-Cell Activating Factor
  • Fish Proteins