| A new endocrine tissue specific gene was isolated and identified, which was located on X chromosome q26.2, encoded a protein containing E2F_TDP domain, highly similar to DP1 and DP2 genes of E2F family, named as DP3. dp3 gene was amplified from pituitary tissue with RT-PCR, and cloned into pcDNA3.1-Myc-HisA(-)vector to construct the recombinant mammalian cell expression PLLsmid. Recombinant DP3protein was detected with western blot, ana was located in cytoPLLsmwith immunofluorescenc subcellular analysis. CHO cell line(CHO-AB4) constitutively expressed DP3 protein was selected with stable cell transfection. And the cell cycle of CHO-AB4 was analyzed with the FACS (Fluorescence Activated Cell Sorter, FACS) compared with the cell line not expressed DP3, FACS showed that the CHO-AB4 had more S-Phase fraction than the negative control, which indicated the dp3 gene might improve the shift from Gl-Phase to S-Phase. Recombinant dp3 pGEX pasmid vector (pGEX-4T-1/dp3-1) was also constructed. The recombinant protein was induced with 0.2mM IPTG at 30? for 4 hours, purified to immunize the rabbit for the DP3 specific antibody. And the high specificity and titre of antibody was detected with ELISA and western blot. |
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