Cloning And Prokaryotic Expression Of P53 Gene And Preliminary Study Of Its Function

Objective To express and purify pET- p53 fusion protein and investigate the effects of the protein on the proliferation of Human leukemia cell line K562 and Human hepatocellular carcinoma cell line SMMC-7721 in vitro. Methods The fragment of human wild type p53 cDNA was amplified by PCR and the expression plasmid of pET-p53 was constructed. Recombinant plasmids were transformed into E.coli BL21,then induced by IPTG at 1mmol/L. The protein was purified by the column of Ni-NAT and analyzed by SDS-PAGE. After treated with fluorescent magnetic nanoparticles(FMNP) modified P53 protein with different concentrations, the proliferation of K562 were tested by MTT assay, clone formation and FCM. We also used laser scanning Confocal microscope (LSCM) to observe the distribution of fluorescent magnetic nanoparticles modified P53 fusion protein in cells. Results Prokaryotic expression vectors of pET- p53 were constructed correctly. pET- p53 fusion protein were successfully expressed and purified. With its increasing concentrations, modified P53 fusion protein reversed its effect on tumor cells significantly. The ratio of inhibition was linear relation to the concentration of P53 fusion protein when concentration of the protein was between 0.1μg/ml and 100μg/ml. Its IC50 was 6.74μg/ml in K562 cells and was 12.39μg/ml in SMMC-7721 cells. Laser scanning Confocal microscope to observe the distribution found that the protein mainly distributed in the nucleus and a small portion of protein was in the cytoplasm. Conclusions The obtained pET- p53 fusion protein has the biological activity of innate P53 protein and it might induce Human leukemia cell line K562 and Human hepatocellular carcinoma cell line SMMC-7721 apoptosis in vitro.