The Study On Molecmle Mechanism Of NE Cleaved PML-RARα In APL Pathogenesis

PARTⅠTHE STUDY ON COLOCALIZATION OF NLS-RARαAND JTV1 INTRACELLULAR LOCALIZATION IN CELLSObjective: To explore colocalization of NLS-RARαand JTV1 in mammalian cell through indirect immunofluorescence technique and laser- confocal microscopy.Methods: HA-tagged fusion protein (pCMV-HA-NLS-RARα) expression vector and Myc-tagged fusion protein (pCMV-Myc-JTV1) expression vector were respectively constructed, identified and transfected into human embryo kidney 293(HEK293) cells. The colocalization of NLS-RARαand JTV1 was observed by indirect immunofluorescence technique. Results: Double restriction enzyme digestion show that pCMV-HA-NLS-RARαand pCMV-Myc-JTV1 were successfully constructed. When HA-NLS-RARαwas tagging by anti-HA polyclonal antibody and Cy3-conjugated goat anti-rabbit IgG,Myc-JTV1 was marking by anti-Myc monoclonal antibody and FITC-conjugated goat anti-mouse IgG, the intracellular colocalization of NLS-RARαand JTV1 was detected by indirect immunofluorescence technique.Conclusion: The bait expression vector of was constructed successfully. HA-NLS-RARαprotein and Myc-JTV1 protein, their intracellular localization in nucleus was analyzed using indirect immunofluorescence, is implying that there is possible interaction between the two proteins in nucleus.PARTⅡConstruction, expression and identification of bait vector of MUT-PML and the two structural domain of MUT-PMLObjective: To investigate the functions of mut-PML and the two structural domains of mut-PML, one domain with ring finger/B-BOX structure and the other one with coiled-coil structure, construct bait expression vector of mut-PML and the two domains for screening the target proteins interacting with the bait protein through the yeast two-hybrid system.Methods: The fragments of mut-PML and the two structural domain of mut-PML were amplified by PCR, and then were cloned into the bait expression vector pGBKT7. After being verified by sequencing, the bait vectors were transformed into AH109 yeast strain. Toxicity, leakage and self-activation of the bait proteins were detected. The expression of the bait protein was analyzed by Western blot.Results: The fragments of mut-PML and the two structural domain of mut-PML were amplified and cloned into pGBKT7 successfully. The bait vectors were transformed into AH109, the bait proteins,BD-mut-PML and BD-PML-B have no toxicity but leakage and self-activation were found, the other one, BD-PML-C has no toxicity, leakage and self-activation. The expression of the bait protein was confirmed by Western blot.Conclusion: The domain of mut-PML with ring finger/B-BOX structure has activity of transcription, the transcription activity of full-length mut-PML is due to the domain. The domain of mut-PML with coiled-coil structure was constructed successfully, which layed the foundations for screening target proteins interacting with the bait protein using the yeast two-hybrid technique. PARTШSCREENING TARGET PROTEINS INTERACTING WITH PML-C BY YEAST TWO-HYBRID SYSTEMObjective: To screen the protein interacting with the domain of PML with coiled-coil structure (PML-C) via the yeast two-hybrid technique, it helps to find out the target proteins interacted with PML-C and further to study the biological function and mechanism of action.Methods: The bait vector of pGBKT7- PML-C was constructed for screening the proteins interacting with PML-C in the leukemic cell cDNA expression library via yeast two-hybrid technique.Results: 43 proteins were screened interacting with PML-C by yeast two-hybrid technique, nine positive clones were identified by retransformation in yeast.Conclusion: There are some proteins interacting with PML-C in cell. The pathogenesis mediated by NE of leukemia maybe related to the biological function altered by the certain protein-protein interaction.