| Objective : To obtain the full length cDNA of platelet-derived growth factor C in Gekko japonicus, and analyze the preliminary characterization of this gene. The expression patterns of PDGF-C in the normal and regenerated spinal cord after tail amputation were investigated as well. To observe the cellular localization of PDGF-C in cells by eukaryotic transfection, and then construct the protokaryon expression vector.Methods : Using the technique of RACE (Rapid Amplification of cDNA Ends ) to obtain the full length cDNA of gPDGF-C from gecko spinal cord, and Northern blot was applied to investigate the size of its transcript and expression pattern in different tissues Using immunochemistry to observe the expression of gPDGF-C protein in normal gecko spinal cord. The expression and location of gPDGF-C in the proximal spinal cord was compared at different time points after tail amputation of gecko by RT-PCR, Western blot and in situ hybridization (ISH). We constructed three different plasmids coding different domain of PDGF-C to observe the change of cellular localization of PDGF-C in gecko cell by eukaryotic transfection. The construct of PDGF-C with GST tag was expressed in prokaryotic cell, and detected by SDS-PAGE electrophoresis. Results :(1) gPDGF-C mRNA spans 2,793 bp, codes 345 amino acids. Aligning gPDGF-C sequence with that of other species, we found the gPDGF-C protein shares 80-88% amino acid identity with the other species. And there was a signal peptide with a putative peptidase cleavage site between residues 22 and 23. Northern blot results showed that PDGF-C gene was widespread expressed in normal gecko, and had only one transcript with 2.8kb. It has a high expression level in heart, lung, kidney and ovary, while a lower level in brain, spinal cord and liver.(2) The result of immunochemistry revealed that the positive signals maily located in gray matter of normal spinal cord, with more positive stained cells in anterior horn than in posterior horn, and a small quantity of positve cells was observed around central canal as well. Double labeling with neuron specific enolase(NSE) suggested some cells with positive signal of PDGF-C were neurons.(3) In situ hybridization showed that the PDGF-C positive signal was observed mainly in gray matter of normal spinal cord with more location in anterior horn than posterior horn, while a small quantity of signal was observed in white matter, and cells around central canal were positively stained as well. After tail amputation, the expression of gPDGF-C was increased at the time of 1d in the proximal stump of spinal cord, and then decreased after 7d.(4) RT-PCR and Western blot results showed that gPDGF-C presented in the normal animals, remarkably up-regulated at 1d after tail amputation, and then gradually recovered till 2w.(5) Transfecting assay with different recombinant construct coding different domain of PDGF-C showed that pEGFP-N3- PDGF-C-FL totally located in cytoplasm and pEGFP-N3- PDGF-C-CD mainly located in nucleus, while pEGFP-N3- PDGF-C-NLS del located both in cytoplasm and in nucleus. It suggested that there was a nuclear localization signal in the carboxyl terminal of gPDGF-C.(6) The recombinant of GST-PDGF-C-ORF plasmid was constructed successfully and the fusion protein was expressed effectively in E. coil.Conclusion :(1) The full length transcript of gPDGF-C was obtained in this study.(2) The PDGF-C gene was widespread expressed in normal gecko.(3) In vivo, gPDGF-C expression was up-regulated in proximate spinal cord after tail amputation.(4) The carboxyl terminal of gecko PDGF-C exists a segment of nuclear localization signal.(5) The recombinant gPDGF-C protein was expressed successfully in prokaryotic cells.
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