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 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>