Xu Z Y, Nie P, Chang M X, Sun B J
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, People's Republic of China.
Fish Shellfish Immunol. 2008 Jun;24(6):701-14. doi: 10.1016/j.fsi.2007.11.019. Epub 2007 Dec 7.
SIMP (source of immunodominant MHC-associated peptides) plays a key role in N-linked glycosylation with the active site of oligosaccharyltransferase, being the source of MHC-peptides in the MHC I presentation pathway. In the present study, the SIMP gene has been cloned from grass carp Ctenopharyngodon idella by rapid amplification of cDNA ends (RACE). The full length of the cDNA sequence is 4384bp, including a 1117bp 5' UTR (untranslated region), a 2418bp open reading frame, and a 849bp 3' UTR. The deduced amino acids of the grass carp SIMP (gcSIMP) are a highly conserved protein with a STT3 domain and 11 transmembrane regions. The gcSIMP spans over more than 24,212bp in length, containing 16 exons and 15 introns. Most encoding exons, except the first and the 15th, have the same length as those in human and mouse. The gcSIMP promoter contains many putative transcription factor binding sites, such as Oct-1, GCN4, YY1, Sp1, Palpha, TBP, GATA-1, C/EBP beta, and five C/EBP alpha binding sites. The mRNA expression of gcSIMP in different organs was examined by real-time PCR. The gcSIMP was distributed in all the organs examined, with the highest level in brain, followed by the level in the heart, liver, gill, trunk kidney, muscle, head kidney, thymus, and the lowest level in spleen. Furthermore, the recombinant gcSIMP has been constructed successfully and expressed in Escherichia coli by using pQE-40 vector, and the polyclonal antibody for rabbit has been successfully obtained, which was verified to be specific. Identification of gcSIMP will help to explore the function in fish innate immunity.
免疫显性MHC相关肽的来源（SIMP）在与寡糖基转移酶活性位点的N-连接糖基化中起关键作用，是MHC I呈递途径中MHC肽的来源。在本研究中，通过cDNA末端快速扩增（RACE）从草鱼（Ctenopharyngodon idella）中克隆了SIMP基因。cDNA序列全长4384bp，包括1117bp的5'非翻译区（UTR）、2418bp的开放阅读框和849bp的3'UTR。推导的草鱼SIMP（gcSIMP）氨基酸序列是一种高度保守的蛋白质，具有一个STT3结构域和11个跨膜区。gcSIMP全长超过24212bp，包含16个外显子和15个内含子。除了第一个和第15个外显子，大多数编码外显子的长度与人和小鼠中的相同。gcSIMP启动子包含许多推定的转录因子结合位点，如Oct-1、GCN4、YY1、Sp1、Palpha、TBP、GATA-1、C/EBPβ以及五个C/EBPα结合位点。通过实时PCR检测了gcSIMP在不同器官中的mRNA表达。gcSIMP分布于所有检测的器官中，在脑中表达水平最高，其次是心脏、肝脏、鳃、躯干肾、肌肉、头肾、胸腺，在脾脏中表达水平最低。此外，已成功构建重组gcSIMP并利用pQE-40载体在大肠杆菌中表达，成功获得了兔多克隆抗体，经验证具有特异性。鉴定gcSIMP将有助于探索其在鱼类先天免疫中的功能。
