[The colocalized relationship between p53 mutants 249M, 273H and hsp70 in HepG2 cells]

To synthesize p53 mutants 249M, 273H with site-directed mutagenesis and construct the relative GFP expression vectors, transfect HepG2 cells and then detect the co-localization of p53 and hsp70.Two sets of primers were designed according to the gene sequence of p53, and mismatches were introduced into primers. Mutagenesis was performed in a two-step PCR. The amplified fragments containing the mutation site from the second PCR were subcloned into the pEGFP-C3 vectors. The localization of p53 and hsp70 was observed in transfected HepG2 cells using confocal microscopy.The sequencing analysis showed that the mutated sites were correct. The expression vectors were constructed successfully. Endogenous hsp70 co-localized in cytoplasm with 273H p53, wt p53 and 249M p53 localized in nucleus.We have found p53 and hsp70 positive products were colocalized in nuclei of human HCC, while the endogenous hsp70 were colocalized in cytoplasm with 273H p53 in HepG2 cells. This contradictory phenomenon suggests that perhaps there are unclear mechanisms existed between the relationship of hsp70 and p53.