The Study On Screening Target Proteins Of Dexamethasone Derivate In Vivio Cells

PART1CONSTRUCTION AND IDENTIFICATION OF THE BAIT EXPRESSION VECTOR OF GLUCOCORTICOID RECEPTOR BINDING DOMAINObjective: To construct the bait expression plasmid pGBKT7-GRα-LBD of glucocorticoid receptor 1igand binding domain, which lay the foundations for constructing small molecule ligand yeast three-hybrid system.Methods: The fragments of GRα-LBD of human K562 cell were amplified by RT-PCR, and then were cloned into the bait expression vector pGBKT7. After being verified by sequencing, the bait vector pGBKT7-GRα-LBD was transformed into AH109 yeast cells. And the expression of the bait protein was analyzed by western blot. Toxicity,leakage and self-activation of the bait protein were detected simultaneously. Results: GRα-LBD was amplified and cloned into pGBKT7 successfully. The bait vector was transformed into AH109 yeast cells successfully and no toxicity, leakage and self-activation were found.The expression of the bait protein was confirmed by western blot.Conclusion: The bait expression vector pGBKT7-GRα-LBD was constructed successfully, which lay the foundations for constructing small molecule ligand yeast three-hybrid system.PART2AMPLI FICATION, PURIFICATION, IDENTIFICATION OF HUMAN K562 CELL cDNA LIBRARY AND ITS TRANSFORMATION INTO YEAST CELLObjective: To ampliy,purify and identify the cDNA library of K562 cell in Eoli DH5αand to transform it into yeast cell for subsequent target protein screening.Methods: The cDNA library of human K562 cell was amplified.Then the library plasmids were extracted and transformed into Y187 yeast cell.The results of transformation were verified by PCR and restriction enzyme analysis. Results: The cDNA library of human K562 cell was successfully amplified and the diversities were verificated. The cDNA library of human K562 cell was also successfully transformed into Y187 yeast cell.Conclusion: Successfully amplification,purification and identification of the cDNA library of human K562 cell which lay the foundations for subsequent protein screening.PART3SCREENING TARGET PROTEINS OF DEXAMETHASONE DERIVATE BY YEAST THREE-HYBRID SYSTEMObjective: Screening target proteins of dexamethasone derivate in K562 cell cDNA library by yeast three-hybrid system.Methods: Yeast strain AH109 containing bait plasmid pGBKT7-GRα-LBD, yeast strain Y187 transformed by K562 cell cDNA library and 0.4μg/ml dexamethasone derivate were mated together,spread the mating product to SD/-Trp/-Leu/-His dropout plate, then pick clones from SD/-Trp/-Leu/-His dropout plate to SD/-Trp/-Leu/-His/-Ade/X-α-gal dropout plate for further nutrition dropout screening.Blue clones were presumed positive clones after 3 times nutrition dropout screening.Yeast plasmids were extracted and transformed into Ecoi DH5α,subsequently plasmids of Ecoi DH5αwere extracted and library fragments were amplifed by PCR and amplification products were digested by only one restrict enzyme. Library plasmids were sorted by PCR and restrict enzyme analysis. We chosen one from every sort randomly as representation to verity weather it was really positive by yeast retransformation test. Positive plasmids were analysised by DNA sequencing. Target protein property was described by analysising sequencing results subsequently.Results: We sequenced forty-three clones at all, and six clones were duplication of the total, in fact we screened thirty-seven different proteins in our experiment. Yeast retransformation test showed that there were twenty politive clones and one new protein among these clones. Yeast mating test were performed of six clones from those that we had screened. Only DDX28 and RanBP9/RanBPM were target proteins interacting with dexamethasone derivate.Conclusion: We screened two target proteins(DDX28 and RanBP9/RanBPM) interacting with dexamethasone derivate. It may be as one envidence on study of dexamethasone derivate anticancer molecular mechanism.