title: Molecular cloning and characterization of TNFSF14 (LIGHT) and its receptor TNFRSF14 (HVEM) in guinea pig (Cavia porcellus).
abstract: 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.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Chunlan
authors.initials: C
authors.lastname: Li
authors.forename: Shanshan
authors.initials: S
authors.lastname: Chen
authors.forename: Jinyun
authors.initials: J
authors.lastname: Song
authors.forename: Hongyan
authors.initials: H
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Gu
authors.forename: Hongxin
authors.initials: H
authors.lastname: Ai
authors.forename: Bo
authors.initials: B
authors.lastname: Zhao
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
mesh_terms: 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 / Q000235:genetics; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D053240:Receptors, Tumor Necrosis Factor, Member 14 / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D013601:T-Lymphocytes / Q000378:metabolism; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D053240:Receptors, Tumor Necrosis Factor, Member 14; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14
keywords: 3D structure; APC; B and T lymphocyte attenuator; BTLA; CRD; Confocal laser; DC; DS; DcR3; Discovery Studio; FBS; GAPDH; HSV; HVEM; Herpes simplex virus; IPTG; LB; LIGHT; LSCM; LTβR; Luria-Bertani (medium); NK; PBS; Phylogenetic analysis; RPE; RT-PCR; SDS-PAGE; SHP; SUMO; SUMO-gpsLIGHT; Small Ubiquitin-like Modifier; T cell receptor; TCR; THD; TNF; TNF homology domain; antigen-presenting cell; base pair(s); bp; cDNA; 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; herpesvirus entry mediator; isoelectric point; kDa; kilodalton(s); laser scanning confocal microscopy; 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; qPCR; real-time quantitative PCR; red fluorescence signal; reverse transcription polymerase chain reaction; sodium dodecyl sulfate-polyacrylamide electrophoresis; sodium hypophosphite; tumor necrosis factor