The Preliminary Study About The Interaction Between Sedlin And Depp

Objective To investigate the interaction between Sedlin and Depp and define the interaction region of Sedlin for Depp by yeast two-hybrid technique. To construct the recombinant expression vector pCDGFP-DEPP. Then COS7 cells were transfected with GFP-tagged DEPP to investigate the localization of Depp.Methods PCR method was used to amplify SEDL-N fragment from pOFF-SEDL plasmid. Both the product and the plasmid pOFF were digested by the same restriction enzymes and then ligated to create a deletion mutant expression vector pOFF-SEDL-N. Both the pOFF-SEDL-N and the plasmid pAS2-1 were digested by the same restriction enzymes and then ligated to creat a deletion mutant expression vector pAS-SEDL-N. Yeast Y190 cells were cotransformed with four groups of plasmids, including pACT2/pAS-SEDL,pACT2/pAS-SEDL-N,pACT2-DEPP/pAS-SEDL and pACT2-DEPP/pAS-SEDL-N, then detect the activity ofβ-galactosidase by Clony-lift Filter Assay. The blue clones were positive, which shows protein-protein interactation. In addition, PCR method was used to amplify the full length cDNA fragment of DEPP from pACT2-DEPP plasmid. And the product was ligated into the T vector to get the recombinant plasmid pUCm-T- DEPP. The recombinant plasmid was digested by the restriction enzymes, and the gene DEPP sequence was inserted into pCDGFP to contruct expression vector pCDGFP-DEPP, which was confirmed by DNA sequencing. Then COS7 cells were transfected with GFP-tagged DEPP to investigate the location of Depp.Results After four groups of plasmids cotransformed into yeast competent cells, assay ofβ-galactosidase activity showed that only the yeast cells transformed with pACT2-DEPP/pAS-SEDL were positive. Restriction endonucleases mapping analysis and DNA sequencing demonstrated that the expression vector encoding Depp was constructed successfully. In COS7 cells, Depp was extensively located both in the nucleus and cytoplasm, but stronger slightly in the nucleus.Conclusions Successfully used yeast-two-hybrid system to identify the interactation between Depp and Sedlin .The C-terminal 27 residues of Sedlin were critical for interaction with Depp. The expression vector pCDGFP-DEPP was constructed successfully. In COS7 cells, Depp was extensively located both in the nucleus and cytoplasm, but stronger slightly in the nucleus.