Fugu Obscurus And Guinea Pigs LIGHT And Its Receptors HVEM Research Cloning, Expression And Function

LIGHT is the14th member of tumor necrosis factor (TNF) cytokine family (TNFSF14). It plays important roles in con-stimulate of T lymphocyte, reducing the apotosis of tumour, autoimmune diseases, graft-versus-host disease (GVHD) and anti-tumor immune. In the present study, a LIGHT homologue has been identified in mefugu{Takifugu obscures), and its biological activities have been characterized. The open reading frame (ORF) of the mefugu LIGHT (designated fLIGHT) covers765bp encoding254amino acids, with a153-aa mature peptide. The predicted three-dimensional (3D) structure of the soluble part of fLIGHT (fsLIGHT) analyzed by "comparative protein modelling" revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that fLIGHT was predominantly expressed in mefugu immune tissues. The SUMO-fsLIGHT/GFP-fsLIGHT were efficiently expressed in Escherichia coli BL21(DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that fsLIGHT could bind to its receptors on mouse lymphocytes. In vitro, the Methyl thiazolyl tetrazolium (MTT) assays analysis indicated that SUMO-fsLIGHT could promote the proliferation of mouse splenic T cells together with anti-mouse CD3. These findings indicate LIGHT may be a potential immunologic factor for enhancing immunological efficacy in fish, and the present findings may provide valuable information for research into the immune system of the mefugu.HVEM, a receptor of LIGHT, is a member of the TNF receptor family. It plays important biological roles in tumor immunity, inflammation, autoimmune diseases occur and thymic negative selection process. Given the imporatance roles in immunoregulation, in this study, experimental animal guinea pig (Cavia porcellus) was selected as the research object. The LIGHT gene of726bp and HVEM of861bp were amplified from guinea pig spleen by RT-PCR. Series of bioinformatics analyses were conducted including the distribution of the encoding gene in the genome, the analyses of the protein three-dimensional structures, the predicted binding sites and the homologous relationship analysis with other species. The results of real-time PCR revealed that both guinea pig LIGHT (gpLIGHT) and guinea pig HVEM (gpHVEM) are expressed in various tissues and elevated in immune organs. The SUMO-gpsLIGHT was efficiently expressed in Escherichia coli BL21(DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that gpsLIGHT could bind to its receptors on guinea pig lymphocytes and mouse T cells. Our findings provide a foundation for further study of the function of LIGHT and HVEM in this important experimental animal.