Cloning, eukaryotic expression and spatial expression patterns of porcine MNSFβ
Received date: 2013-05-11
Revised date: 2013-07-29
Online published: 2013-11-25
MNSFβ(Monoclonal nonspecific suppressor factor β) is a natural immunosuppressive factor which has been reported to be involved in various biological processes, such as immune responses, cell division, stress response, cell apoptosis, and nuclear transport. However, study on porcine MNSFβ has been rarely reported. In this study, the full-length sequence 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 microscopy, 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 MNSFβ protein was a stable protein consisting of a ubiquitin-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 MNSFβ is highly con-served in the process of evolution. Fluorescence signal showed that the fusion protein GFP-MNSFβ was 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.
WANG Jing-Na, JIANG Peng-Fei, KANG Zhan-Zhan, LI Deng-Yun, HUA Lin-Lin, ZHANG Yang, ZHANG Shu-Xia, ZHANG De-Li . Cloning, eukaryotic expression and spatial expression patterns of porcine MNSFβ[J]. Hereditas(Beijing), 2013 , 35(12) : 1377 -1383 . DOI: 10.3724/SP.J.1005.2013.01377
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