Banded Grouper B Lymphocyte Stimulator (BAFF) And Zebrafish Cloning TNFSF14, Expression And Biological Function

B cell activating factor (BAFF) and lymphotoxin-like, exhibits inducible expression, and competes with HSV glycopmtein D (gD) for HVEM, a receptor expressed by T lymphocytes (LIGHT) are two important members of the TNF ligand superfamily. BAFF extensive participate in regulating peripheral B lymphocytes activation, proliferation, and differentiation, LIGHT can provide costimulatory signals to T cells and induce the apoptosis of some tumor cell lines.They play important role in lymphoid organogenesis, auto-immunity diseases and antiviral immunity. The study of yellow grouper (Epinephelus awoara) BAFF and Zebrafish (Danio rerio) LIGHT at home and abroad is still blank.1-. The molecular cloning, expression and functional analysis of TNFSF13b (BAFF) in yellow grouper, Epinephelus awoara.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 was1442bp and contained an open reading frame of780bp encoding a putative protein of259amino acids. Amino acids sequence comparison indicated that EaBAFF possessed the TNF signature. a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the soluble part of gBAFF (gsBAFF) analyzed by "comparative protein modelling" revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that gBAFF was predominantly expressed in goat lymphoid tissue spleen. 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-8assay 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.2、Isolation and characterization of LIGHT (TNFSF14) gene homologue in zebrafish(Danio rerio). The full-length open reading frame (ORF) of zLIGHT cDNA has been cloned from zebrafish Danio rerio using RT-PCR. The full-length open reading frame (ORF) of zLIGHT cDNA consists of708bp encoding a protein of235amino acids. Real-time quantitative PCR (qPCR) analysis suggested that zLIGHT was predominantly expressed in zebrafish spleen. The soluble LIGHT (zsLIGHT) had been cloned into the pSUMO vector, SDS-PAGE and Western blotting analysis confirmed that the recombinant protein SUMO-zsLIGHT was efficiently expressed in Escherichia coli BL21(DE3). Laser scanning confocal microscopy analysis showed that SUMO-zsLIGHT could bind to its receptors on T cells. Zebrafish may conduct as a model animal for further research on LIGHT.