Site-directed mutagenesis and construction of expression vector containing human p53 mutants 175 H,248 W and 273 H

Objective To synthesize p53 mutants 175 H,248 W and 273 H with site-directed mutagenesis and to construct the relative expression vectors.Methods 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 pcDNA3.1 vector.Results The sequencing analysis showed that the mutated sites were correct,the sites of 175,248 and 273 were changed from Arg to His, Arg to Try,Arg to His respectively,suggesting that the expression vectors were constructed successfully.Conclusion PCR site-directed mutagenesis method is accurate and highly efficient,and the construction of expression vector containing 175 H,248 W and 273 H of p53 gene provides a solid foundation for further studies.