| Tumor necrosis factor alpha (TNF-α), a pleiotropic cytokine with a wide biological activities, exists as a 26kDa transmembrane form (transmembrane TNF-α, mTNF-α) and a 17kDa soluble form (secretory TNF-α, sTNF-α), and is produced by T cells, monocytes/macrophages and NK cells. mTNF-αis the precursor of sTNF-αand can be cleaved into sTNF-αby TNF-αconverting enzyme (TACE).It was found by recent studies that mTNF-αnot only as ligand functions on TNF receptor bearing target cell via"forward signaling", but also as receptor on TNF-αproducing cell through"reverse signaling".Our previous work confirmed that the TRAF1 could be co-precipitated with mTNF-α, suggesting that this signal molecule may interact with the cytoplasmic segment of mTNF-αdirectly or indirectly to transduce reverse signaling.The distinction between the primary structure of mTNF-αand sTNF-αis the signal peptide of mTNF-α, containing 76 amino acids, which is consisted of three segments: linker, transmembrane and cytoplasmic fragment.Tumor necrosis factor receptor associated factors (TRAFs) are a major group of intracellular adaptors that bind to many family members of the TNF receptor directly or indirectly. So far, six mammalian TRAFs, TRAF1 through TRAF6, have been identified. They can induce the activation of several kinase cascades that ultimately lead to the activation of signal transduction pathways such as NF-κB, JNK etc, which can regulate cellular processes ranging from cell proliferation and differentiation to apoptosis. In this study, the TNF-SP-EGFP fusion protein was constructed and was expressed in COS-7 cell line with transient transfection. The location of TNF-SP-EGFP fusion protein was observed through confocal microscope, the interaction between TNF-SP and TRAF1 was also determined through the co-localization of confocal microscope, suggesting that TNF-SP might interact with TRAF1 through N-terminal of TRAF1. The aim of this study is to provide clues for further study of the signal transduction pathways of the reverse signaling. The major results in the present study are as follows:一. Construction, cloning and identification of TNF-SP-EGFP1. Construction and cloning of TNF-SP-EGFP recombinants: Using the plasmid TNF-SP-PcDNA3.0 as template, the TNF-SP gene was amplified by PCR. After digestion with endonucleases, the TNF-SP fragment was inserted into EGFP plasmid at EcoRI and BamHI by T4 DNA ligase. Then the TNF-SP-EGFP recombinant was transformed into E. coli DH5αand the positive clones were screened by Kanamycin resistance.2. Identification of positive clones: The positive clones were confirmed firstly by colony PCR, followed by further identification with endonucleases digestion, showing the cleaved TNF-SP fragment from the recombinant with the molecule weights of 228bp, DNA sequence analysis also proved that the recombinant contains sequences encoding TNF-SP without mutations, suggesting TNF-SP-EGFP expressing plasmid was successfully constructed.二.Eukaryotic expression of TNF-SP-EGFP and the subcellular location of the fusion protein1. The transient expression and subcellular localization of the TNF-SP-EGFP fusion proteins in the transfected COS-7 cells was analyzed by fluorescent microscopy and confocal microscopy. And, confocal microscopy observation showed that the TNF-SP-EGFP was distributed in cytoplasm.2. The recombinant of TNF-SP-EGFP was cotransfected with TRAF1-pDsRedN1 into COS-7 cells. Fluorescent microscopy observation showed there was yellow fluorescence in the cells cotransfected with TNF-SP-EGFP (green fluorescence) and TRAF1-pDsRedN1 (red fluorescence), the result indicated that TNF-SP probably bind with the N-terminal of the TRAF1.In summary, the express plasmid of TNF-SP-EGFP was successfully constructed and TNF-SP-EGFP fusion protein was expressed by eukaryotic cells. The cellular localization of TNF-SP was confirmed in cytoplasm. And probably TNF-SP binds with the N-terminal of the TRAF1. These results provide an effective research tool and detection means to further study the interactions between TNF-SP and TRAF1 signal molecules.
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