Molecular cloning and characterization of TNFSF14 (LIGHT) and its receptor TNFRSF14 (HVEM) in guinea pig (Cavia porcellus).
LIGHT (lymphotoxin-related inducible ligand that competes with herpes simplex virus (HSV) glycoprotein D for herpesvirus entry mediator on T cells) is a member of the tumor necrosis factor (TNF) ligand superfamily, which plays important roles in inflammatory and immune responses. In the present study, the cDNAs of guinea pig (Cavia porcellus) LIGHT (designated as gpLIGHT) and its receptor herpes virus entry mediator (designated as gpHVEM) were amplified from spleen by reverse transcription polymerase chain reaction (RT-PCR). The ORFs of gpLIGHT and gpHVEM cover 726 and 861 bp, encoding predicted proteins with 241 and 286 aas, respectively. The three-dimensional (3D) structure, phylogenetic relationships, and characterization of both genes were also analyzed. We also generated a 3D model to verify interaction between the two proteins. Real-time quantitative PCR (qPCR) analysis revealed that both LIGHT and HVEM are constitutively expressed in guinea pig various tissues. A fusion protein SUMO (Small Ubiquitin-like Modifier)-gpsLIGHT (the soluble mature part of gpLIGHT) was efficiently expressed in Escherichia coli BL21 (DE3) and purified using metal chelate affinity chromatography (Ni-NTA). Laser scanning confocal microscopy (LSCM) showed that gpsLIGHT can bind its receptors on T cells. The LIGHT-HVEM signaling pathway plays an important role in the immune system, and our results might provide a platform for further research into the effects of LIGHT and HVEM.
['Li|Chunlan|C|', 'Chen|Shanshan|S|', 'Song|Jinyun|J|', 'Liu|Hongyan|H|', 'Gu|Wei|W|', 'Ai|Hongxin|H|', 'Zhao|Bo|B|', 'Zhang|Shuangquan|S|']
D000595:Amino Acid Sequence D000818:Animals D001483:Base Sequence D001665:Binding Sites D002460:Cell Line D003001:Cloning, Molecular D015870:Gene Expression D023061:Gene Order D006168:Guinea Pigs D006801:Humans D051379:Mice D008958:Models, Molecular D008969:Molecular Sequence Data D009928:Organ Specificity D010802:Phylogeny D011485:Protein Binding D011487:Protein Conformation D053240:Receptors, Tumor Necrosis Factor, Member 14 D016415:Sequence Alignment D013601:T-Lymphocytes D053326:Tumor Necrosis Factor Ligand Superfamily Member 14
LIGHT HVEM Herpes simplex virus HSV herpesvirus entry mediator tumor necrosis factor TNF cDNA reverse transcription polymerase chain reaction RT-PCR bp real-time quantitative PCR qPCR SUMO laser scanning confocal microscopy LSCM 3D structure APC B and T lymphocyte attenuator BTLA CRD Confocal laser DC DS DcR3 Discovery Studio FBS GAPDH IPTG LB LTβR Luria-Bertani (medium) NK PBS Phylogenetic analysis RPE SDS-PAGE SHP SUMO-gpsLIGHT Small Ubiquitin-like Modifier T cell receptor TCR THD TNF homology domain antigen-presenting cell base pair(s) cDNA cloning complementary DNA cysteine-rich domain dNTP decoy receptor 3 dendritic cell deoxyribonucleoside triphosphate fetal bovine serum final isopropyl-beta-D-thiogalactopyranoside glyceraldehyde-3-phosphate dehydrogenase isoelectric point kDa kilodalton(s) lymphotoxin homolog, inducible and competes with HSV glycoprotein D for HveA and is expressed on T lymphocytes lymphotoxin β receptor natural killer pI phosphate-buffered saline red fluorescence signal sodium dodecyl sulfate-polyacrylamide electrophoresis sodium hypophosphite