Construction of pNTAP-MK2 eukaryotic expression plasmid and establishment of a cell line for its stable expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct pNTAP-MK2 eukaryotic expression plasmid and establish a HEK293 cell line stably expressing tandem affinity purification (TAP)-tagged MK2.</p><p><b>METHODS</b>The MK2-encoding region was subcloned into the vector pNTAP to construct the recombinant plasmid pNTAP-MK2, which was subsequently transformed into DH5 alpha.E.coli. After identification by PCR, digestion with restriction endonuclease and sequencing, the recombinant expression plasmid was transfected into HEK293 cells via liposome, and the cell line with stable expression of exogenous TAP tag-MK2 gene was selected by antibiotic G418. The expression and localization of the fusion protein TAP tag-MK2 were detected by Western blotting and immunofluorescence assay.</p><p><b>RESULTS</b>The results of PCR, restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pNTAP-MK2. Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection with G418 selection. Immunofluorescence assay identified the expression product TAP tag-MK2 mainly in the cell nuclei.</p><p><b>CONCLUSION</b>The eukaryotic expression vector pNTAP-MK2 has been successfully constructed, and in the established cell line with stable expression of TAP tag-MK2, TAP tag does not influence the localization of exogenous MK2.</p>

Construction of different mutants of HA-tagged human RAGE gene and their eukaryotic expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct eukaryotic expression vectors for HA-tagged receptor for advanced glycation end products (RAGE) mutants.</p><p><b>METHODS</b>Site-directed mutagenesis was applied to wild-type RAGE gene cloned in the pcDNA3 vector with HA tag to obtain the mutants pcDNA3-HA-RAGE(S391A), pcDNA3-HA-RAGE(S399A), pcDNA3-HA-RAGE(S400A), and pcDNA3-HA-RAGE(T401A). After identification by sequencing, the mutants were transfected into HEK293 cells, and the expression of these mutants were detected by Western blotting using anti-HA antibody.</p><p><b>RESULTS</b>The HA-tagged RAGE mutants constructed were verified successfully by sequencing, and highly expressed in HEK293 cells.</p><p><b>CONCLUSION</b>The success in constructing HA-tagged RAGE mutants, which are highly expressed in eukaryotic cells, may facilitate the functional study of RAGE in cell signal transduction.</p>

Construction and expression of different mutants of human p53 and their effects on arsenite-induced cell apoptosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct different mutants of human p53 for expression in eukaryotic cells and investigate the effects of these mutants on stress-induced cell apoptosis.</p><p><b>METHODS</b>Human p53 cDNA was amplified by PCR and cloned into pcDNA3/HA vector following the routine procedures. The Ser15 and Ser46 of p53 were mutated to Ala and identified by enzyme digestion and PCR, and these mutants were expressed in NIH3T3 cells and detected by Western blotting. After transfection with the plasmids of different p53 mutants, the NIH3T3 cells were double-stained with AnnexinV-FITC and propidium iodide for apoptotic analysis using flow cytometry.</p><p><b>RESULTS</b>The recombinant plasmids of HA-tagged wild-type p53, HA-p53(WT), and its mutants, HA-p53(S15A) and HA-p53(S46A), were successfully constructed and expressed efficiently in NIH3T3 cells. The apoptotic ratio of p53(WT)-transfected cells induced by arsenite increased and that of p53(S15A)-transfected cells decreased significantly after arsenite stimulation, but no significant changes occurred in the apoptosis of p53(S46A)-transfected cells.</p><p><b>CONCLUSION</b>The phosphorylation on Ser15 of p53 plays an important role in mediating arsenite-induced cell apoptosis.</p>

Construction and expression of the fusion vector for HA-tagged human RAGE gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a mammalian expression vector for HA-tagged receptor of advanced glycation end products (RAGE).</p><p><b>METHODS</b>Human RAGE cDNA codon region was amplified by PCR from human cDNA library and cloned into the pcDNA3 vector following routine procedures. After identification by enzyme digestion, PCR and sequencing, the correct recombinant vector RAGE/pcDNA3 was inserted with HA sequence behind the signal peptide sequence of RAGE. After identification by sequencing, HA-RAGE/pcDNA3 was transfected into HEK293 cells, and its expression was detected by Western blotting.</p><p><b>RESULTS</b>Identification by enzyme digestion, PCR and sequencing, confirmed the validity of the recombinant vector RAGE/pcDNA3, and HA-RAGE/pcDNA3 was highly expressed in HEK 293 cells.</p><p><b>CONCLUSION</b>HA-tagged RAGE is successfully constructed and expressed in mammalian cells, which may facilitate functional study of RAGE in cell signal transduction.</p>

Effect of p53 gene knockout on cell migration / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of p53 gene in serum-induced cell migration.</p><p><b>METHODS</b>The effects of p53 knockout on serum-induced formation of lamellipodia and cell migration were observed using Transwell cell migration system.</p><p><b>RESULTS</b>p53(+/+) cells developed lamellipodia upon serum stimulation and showed enhanced activity of cell migration, but these effects were not observed in p53 knockout cells after serum stimulation.</p><p><b>CONCLUSION</b>p53 plays a role in serum-induced cell migration.</p>