小鼠NOD2/CARD15基因的克隆、测序及表达分析。

Cloning, sequencing and expression analysis of the mouse NOD2/CARD15 gene.

作者信息

Iwanaga Y, Davey M P, Martin T M, Planck S R, DePriest M L, Baugh M M, Suing C M, Rosenbaum J T

机构信息

Casey Eye Institute, Oregon Health & Science University, Portland, Oregon 97201, USA.

出版信息

Inflamm Res. 2003 Jun;52(6):272-6. doi: 10.1007/s00011-003-1170-z.

DOI:10.1007/s00011-003-1170-z

PMID:12835899

Abstract

BACKGROUND

Mutations in the human NOD2/CARD15 gene have been associated with Crohn's disease and Blau syndrome. The objective of the present study was to clone the murine form of NOD2 and characterize its tissue distribution, function and response to inflammatory stimuli.

METHODS

Murine NOD2 was isolated using anchored polymerize chain reaction (PCR). Sequence analysis confirmed the identification of full-length cDNA representing the murine NOD2 gene. Using this sequence to search a Mus musculus supercontig database, NOD2 genomic DNA was identified. NOD2 was transfected into human embryonic kidney (HEK) cells and nuclear factor kappa B (NF-kappaB) activation was measured using a reporter assay. Tissue distribution and changes in transcription in mouse monocytes in response to inflammatory stimuli was determined by real time PCR.

RESULTS

The NOD2 gene spans 39 KB and contains 12 coding exons on chromosome 8. Expression of mouse NOD2 into HEK cells resulted in NF-kappaB activation. NOD2 was found to be expressed in all mouse tissues analyzed except skin, with highest levels in lung, thymus and spleen. NOD2 mRNA levels increased greater than two-fold in a monocyte cell line in response to lipopolysaccharide, lipoteichoic acid, interferon-g and tumor necrosis factor-alpha.

CONCLUSIONS

Common structural and functional features between human and mouse NOD2 were identified. This should allow for development of relevant animal models to evaluate the role of NOD2 in chronic inflammatory disorders.

摘要

背景

人类NOD2/CARD15基因的突变与克罗恩病和布劳综合征相关。本研究的目的是克隆小鼠形式的NOD2，并对其组织分布、功能及对炎症刺激的反应进行特性分析。

方法

采用锚定聚合酶链反应（PCR）分离小鼠NOD2。序列分析证实了代表小鼠NOD2基因的全长cDNA的鉴定。利用该序列搜索小家鼠重叠群数据库，鉴定出NOD2基因组DNA。将NOD2转染到人胚肾（HEK）细胞中，并使用报告基因检测法测量核因子κB（NF-κB）的激活情况。通过实时PCR确定小鼠单核细胞中NOD2的组织分布及对炎症刺激的转录变化。

结果

NOD2基因跨度为39KB，在8号染色体上包含12个编码外显子。将小鼠NOD2表达于HEK细胞中可导致NF-κB激活。发现NOD2在除皮肤外的所有分析小鼠组织中均有表达，在肺、胸腺和脾脏中表达水平最高。在单核细胞系中，NOD2 mRNA水平在脂多糖、脂磷壁酸、干扰素-γ和肿瘤坏死因子-α刺激下增加超过两倍。