Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Gene. 2013 Sep 10;526(2):374-84. doi: 10.1016/j.gene.2013.05.031. Epub 2013 May 31.
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.
LIGHT（与单纯疱疹病毒（HSV）糖蛋白 D 竞争疱疹病毒进入介质的淋巴毒素相关诱导配体，在 T 细胞上）是肿瘤坏死因子（TNF）配体超家族的成员，在炎症和免疫反应中发挥重要作用。在本研究中，通过逆转录聚合酶链反应（RT-PCR）从脾脏中扩增了豚鼠（Cavia porcellus）LIGHT（命名为 gpLIGHT）及其受体疱疹病毒进入介质（命名为 gpHVEM）的 cDNA。gpLIGHT 和 gpHVEM 的 ORF 分别覆盖 726 和 861 bp，编码预测蛋白分别具有 241 和 286 个氨基酸。还分析了这两个基因的三维（3D）结构、系统发育关系和特征。我们还生成了一个 3D 模型来验证这两种蛋白质之间的相互作用。实时定量 PCR（qPCR）分析显示，LIGHT 和 HVEM 在豚鼠各种组织中均持续表达。通过大肠埃希菌 BL21（DE3）高效表达并使用金属螯合亲和层析（Ni-NTA）纯化了 SUMO（Small Ubiquitin-like Modifier）-gpsLIGHT（gpLIGHT 的可溶性成熟部分）融合蛋白。激光扫描共聚焦显微镜（LSCM）显示 gpsLIGHT 可以与 T 细胞上的受体结合。LIGHT-HVEM 信号通路在免疫系统中起着重要作用，我们的结果可能为进一步研究 LIGHT 和 HVEM 的作用提供一个平台。
