| Tumor necrosis factor alpha (TNF-α), a pleiotropic cytokine with a wide biological activities, exists as a 26kDa transmembrane form (transmembrane TNF-α, TM-TNF-α) and a 17kDa soluble form (secretory TNF-α, S-TNF-α), and is produced by T cells, monocytes/macrophages and NK cells. TM-TNF-αis the precursor of S-TNF-αand can be cleaved into S-TNF-αby TNFαconverting enzyme (TACE).It was found by recent studies that TM-TNF-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 IKK-αand TRAF1 could be co-precipitated with TM-TNF-αrespectively, suggesting that these two signal molecules may interact with the cytoplasmic segment of TM-TNF-αdirectly or indirectly to transduce reverse signaling.The aim of the present study is to obtain the full length of reading frames for IKK-αand TRAF1 and construct recombinants of IKK-α-EGFP and TRAF1- DsRed (EGFP/DsRed are a pair of fluorescent proteins) for Fluorescent Resonant Energy Transfer (FRET) analysis. Meanwhile, a positive plasmid EGFP-pDsRed was also constructed that can be applied to FRET detection as a positive control. We can use these three recombinants to further study the in vivo interactions of the signal molecules in alive cells by TRET detection. The main results in the present study are as follows:⒈Construction, cloning and identification of IKK-α-pEGFP, TRAF1-pDsRed and EGFP-pDsRed⑴Construction and cloning of recombinants: Total RNA from Raji cells was isolated and reversely transcribed into cDNA. Using cDNA as template, the cDNA fragments coding for IKK-αand TRAF1 were amplified by PCR and identified by nest PCR. Meanwhile, the plasmid pIRES-EGFP was used as templates for amplification of EGFP by PCR. After digestion with endonucleases, the IKK-αcDNA fragment was inserted into pEGFP-N3 plasmid at Xho I and EcoR I and the cDNA fragments for TRAF1 and EGFP were inserted into pDsRed-N1 plasmid at EcoR I and SacII respectively by T4 ligase. The IKK-α-EGFP, TRAF1-DsRed and EGFP-DsRed recombinants were transformed into E. coli DH5αand the positive clones were screened by Kana resistance.⑵Identification of positive clones: The positive clones were confirmed firstly by bacteria PCR, followed by further identification with endonucleases digestion, showing cleaved IKK-α, TRAF1 and EGFP fragments from the three recombinants with the molecule weights of 2.2kb, 1.3kb and 720bp respectively. DNA sequence analysis also proved that the three recombinants contains sequences encoding IKK-α, TRAF1 and EGFP without mutations, suggesting IKK-α-pEGFP,TRAF1-pDsRed,EGFP-pDsRed expressing plasmids were successfully constructed.2. Eukaryotic expression of IKK-α-EGFP, TRAF1-DsRed and EGFP-DsRed fusion proteinsThe three recombinants IKK-α-pEGFP, TRAF1-pDsRed and EGFP-pDsRed were transfected into COS7 cells respectively. It was shown that the IKK-α-EGFP fusion protein (green fluorescence) dispersedly distributed at the whole cytoplasm of cells, the TRAF1-DsRed fusion protein (red fluorescence) was localized in compartments of cells. The EGFP-pDsRed fusion protein expressing COS7 cells were stained by both green and red fluorescence and the fusion proteins were mainly localized in the cytoplasm of cells. All of these observations confirmed that the IKK-α-EGFP, TRAF1-DsRed and EGFP-DsRed fusion proteins were successfully expressed by eukaryotic cells.To summarize, the expressing plasmids of IKK-α-pEGFP, TRAF1-pDsRed and EGFP-pDsRed were successfully constructed and IKK-α-EGFP, TRAF1-DsRed and EGFP-DsRed fusion proteins were expressed by eukaryotic cells. The cellular localization of IKK-αand TRAF1 were also confirmed. These recombinants provide an effective research tool and detection means to further study the interactions between TM-TNF-αand these two signal molecules. EGFP-pDsRed plasmid can be also applied for FRET detection as a positive control.
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