Abstract:

Objective    To determine interactions between p65 and SMRT proteins in  the acute myelogenous leukemia cell line (THP-1) by co-immunoprecipitation. Methods    The human p65 gene and SMRT gene were synthesized in vitro and cloned into the pUC57 vector. Corresponding DNA was amplified by PCR and the PCR products were subcloned into eukaryotic  vectors of pCMV-HA and pCMV-Myc after restriction enzyme digestion. Then, these recombined plasmids were confirmed by restriction enzyme digestion and DNA sequencing and co-transfected into THP-1 cell lines. The interactions between p65 and SMRT were detected by co-immunoprecipitation. Results      Restriction enzymes digestion and DNA sequencing showed that the recombinant eukaryotic vectors of pCMV-HA-p65 and pCMV-Myc-SMRT were correctly constructed. After being immunoprecipitated by anti-HA and anti-Myc multi-antibodies，fusion proteins of  HA-p65 and Myc-SMRT were identified by Western blotting with anti-Myc and anti-HA antibodies. Conclusion      Recombinant eukaryotic vectors of pCMV-HA-p65 and pCMV-Myc-SMRT were successfully constructed. The interaction of P65 and SMRT should be identified  by co-immunoprecipitation after co-expression in THP-1 cell line, which can serve as a tool for further study of the pathogenesis of AML in children and targeting therapy of leukemia.

Key words: Co-immunoprecipitation; P65; SMRT; Myelocytic Leukemia