猪免疫抑制因子MNSFβ的克隆、表达与组织分布

Cloning, eukaryotic expression and spatial expression patterns of porcine MNSFβ

MNSFβ(Monoclonal nonspecific suppressor factorβ)是一种天然免疫抑制因子,参与机体免疫反应、应激反应、细胞分裂、细胞凋亡和核转运等生物过程,但其功能在猪中鲜有报道。文章通过电子克隆得到猪MNSFβ的全长序列,利用RT-PCR首次从猪脾脏中克隆了包含完整开放阅读框的MNSFβcDNA(GenBank登录号:KF77642500)片段,测序正确后分析猪MNSFβ的核酸序列和蛋白质序列。将猪MNSFβ基因编码区序列插入表达载体pEGFP-C1,构建真核表达载体pEGFP-MNSFβ;用脂质体将重组质粒转染到猪脐静脉内皮细胞(SUVEC)中,利用荧光检测、Western blot和激光共聚焦分析SUVEC中猪MNSFβ的表达;并用实时荧光定量PCR对猪MNSFβ在不同组织的表达丰度进行分析。结果表明:猪MNSFβ基因全长402 bp,编码133个氨基酸,含一个外显子。生物信息学分析表明,猪MNSFβ为无信号肽的稳定蛋白,由泛素样结构域与核糖体蛋白S30组成。对猪MNSFβ与已发现的18个物种的MNSFβ氨基酸序列进行相似性分析和进化树分析,显示其蛋白相似度均在91%以上,且进化距离都小于0.05,说明MNSFβ在各物种间高度保守。荧光检测和Western blot结果显示,猪MNSFβ在SUVEC中成功表达,激光共聚焦显示其在细胞质与细胞核内均有分布。组织表达谱分析表明,猪MNSFβ在各免疫相关组织广泛表达,但在肺脏中未检测到表达,说明其在机体免疫反应中发挥重要作用。

MNSFβ（Monoclonal nonspecific suppressor factor β） is a natural immunosuppressive factor which has beenreported to be involved in various biological processes, such as immune responses, cell division, stress response, cell apop- tosis, and nuclear transport. However, study on porcine MNSFβ has been rarely reported. In this study, the full-length se- quence of porcine MNSFβ（GenBank accession number： KF77642500） was predicted in silicon and its cDNA sequence was obtained through RT-PCR from porcine spleen. The nucleic acid and protein sequences were analyzed. Then, the gene was subcloned into pEGFP-C1 to construct a recombinant plasmid pEGFP-MNSFβ which was transfected into swine umbilical vein endothelial cells （SUVECs） using Lipofectamine 2000. The expression of GFP was detected by fluorescence micros- copy, Western blot, and laser confocal fluorescence microscopy. The spatial expression patterns of porcine MNSFβ were detected by real-time qPCR. Results showed that the full length of porcine MNSFβ was 402 bp encoding 133 amino acids with only one exon. Bioinformatics analysis showed that porcine MNSFβprotein was a stable protein consisting of a ubiq- uitin-like domain fused to the ribosomal protein S30 with no signal peptide. The analyses of homology and phylogenetic tree of porcine MNSFβ and its homologs in other 18 species showed that the identities of MNSFβ protein sequence were higher than 91% among different species and the evolutionary distance was less than 0.05. It indicates that MNSFβis highly conserved in the process of evolution. Fluorescence signal showed that the fusion protein GFP-MNSFβwas successfully expressed in SUVECs which was then confirmed by Western blot. Laser confocal fluorescence microscopy showed that MNSFβ was expressed in both nucleus and cytoplasm. Analysis of spatial expression patterns showed that procine MNSFβ was widely expressed in immune tissues, but not in lung, suggesting that MNSFβ may play an important role in immune response.