[Cloning, eukaryotic expression and spatial expression patterns of porcine MNSFbeta].

MNSFbeta (Monoclonal nonspecific suppressor factor beta) 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 MNSFbeta has been rarely reported. In this study, the full-length sequence of porcine MNSFbeta (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-MNSFbeta 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 MNSFbeta were detected by real-time qPCR. Results showed that the full length of porcine MNSFbeta was 402 bp encoding 133 amino acids with only one exon. Bioinformatics analysis showed that porcine MNSFbeta 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 MNSFbeta and its homologs in other 18 species showed that the identities of MNSFbeta protein sequence were higher than 91% among different species and the evolutionary distance was less than 0.05. It indicates that MNSFbeta is highly conserved in the process of evolution. Fluorescence signal showed that the fusion protein GFP-MNSFbeta was successfully expressed in SUVECs which was then confirmed by Western blot. Laser confocal fluorescence microscopy showed that MNSFbeta was expressed in both nucleus and cytoplasm. Analysis of spatial expression patterns showed that procine MNSFbeta was widely expressed in immune tissues, but not in lung, suggesting that MNSFbeta may play an important role in immune response.

A potential molecular model for studying apoptosis enhanced by the interaction of BCL-G with JAB1 in swine.

BCL-G, an apoptotic factor in Bcl-2 family, is involved in several kinds of diseases by interacting with several proteins. Although many studies on mouse and human BCL-G have been reported, porcine BCL-G (pBCL-G) has been little investigated. In this study, our results showed that pBCL-G was universally expressed in porcine tissues. The BH2 domain affected the subcellular distribution of pBCL-G protein. pBCL-G could interact with porcine JAB1 (pJAB1), by which its subcellular distribution was affected. pBCL-G promoted staurosporine-induced apoptosis that was significantly enhanced by interaction of pBCL-G with pJAB1. The apoptosis at least partially depended on the activated caspase-8, -9 and -3. Owing to the close phylogenetic distance between pigs and humans and their many physiological similarities, our findings may provide a potential molecular model to study human BCL-G and also may have implications in the treatment of diseases relevant with BCL-G.