The Study On Screening Target Proteins Interacting With NLS-RARα In Vivio Cells

PART 1 CONSTRUCTION AND IDENTIFICATION OF THE BAIT EXPRESSION VECTOR OF NLS-RARαAbstract Objective: To construct the bait expression vector pGBKT7-NLS-RARαof retinoic acid receptor variant protein, for screening the target proteins interacting with the bait protein through the yeast two-hybrid technique.Methods: The fragments of NLS-RARαbinding domain was amplified by PCR, and then was cloned into the bait expression vector pGBKT7. After being verified by sequencing, the bait vector pGBKT7-NLS-RARαwas transformed into AH109 yeast cells. Then the expression of the bait protein was analyzed by Western Blotting. Toxicity and self-activation of the bait protein were detected.Results: NLS-RARαwas amplified and cloned into pGBKT7 successfully. The bait vector was transformed into AH109 as well and no toxicity and self-activation were found. The expression of the bait protein was confirmed by Western Blotting.Conclusion: The bait expression vector of was constructed successfully, which layed the foundations for screening target proteins interacting with the bait protein using the yeast two-hybrid technique.PART 2 SCREENING TARGET PROTEINS INTERACTING WITH NLS-RARαBY YEAST TWO-HYBRID SYSTEMObjective: To screen the protein interacting with Retinoic Acid Receptor variant protein (NLS-RARα) via the yeast two-hybrid technique, it helps to find out the targets protein interacted with NLS-RARαand further to study the biological function.Methods :The bait vector of pGBKT7- NLS-RARαwas constructed for screening the proteins interacting with NLS-RARαin the K562 cell cDNA expression library via yeast two-hybrid technique. Furthermore, the protein-protein interaction was confirmed with re-transformation in yeast.Results : Sixteen proteins were screened by yeast two-hybrid technique. eight positive clones were identified by retransformation in yeast.Conclusions: There are some proteins interacting with NLS-RARαin cell. The pathogenesis of leukemia maybe related to the biological function altered by the certain protein-protein interaction.PART 3 IDENTIFICATION OF INTERACTION BETWEEN NLS-RARαAND JTV-1 BY CO-IMMUNOPRECIPITATION IN VITROObjective: to explore the interaction between NLS-RARαand JTV-1 by co-immunoprecipitation.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 interaction between NLS-RARαand JTV1 was detected by co-immunoprecipitation.Results: Double restriction enzyme digestion show that pCMV-HA-NLS-RAR and pCMV-Myc-JTV1 were successfully constructed.When HA-NLS-RAR was immunoprecipitated by anti-HA polyclonal antibody, Myc-JTV1 was identified by western blotting with anti-Myc monoclonal antibody from immunoprecipitated complex. Conclusion: The recombinated vector pCMV-HA-NLS-RAR and pCMV-Myc-JTV1 were constructed successfully.The interaction between NLS-RAR and JTV1 was identified by co-immunoprecipitation...