Preparation of an Anti-Cdx-2 Antibody for Analysis of Different Species Cdx-2 Binding to acat2 Promoter
SONG Bao-Liang¹, QI Wei^1,2, WANG Can-Hua^1,3, YANG Jin-Bo¹, YANG Xin-Ying¹, LIN Zhi-Xin³, LI Bo-Liang^1*

( ¹State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China;
²Department of Biological Science and Technology, Nanjing University, Nanjing 210093, China;
³ Department of Biological Science and Technology, Shanghai Jiaotong University, Shanghai 200030, China)

Abstract The homeodomain protein, Cdx-2, as transcription factor has been implicated in the transcriptional regulation of genes expressed in small intestine and the process of tumorgenesis. In current work, a conserved mouse Cdx-2 domain (mCdx-2D) coded by its cDNA fragment, which was amplified and cloned into the expression vector pGEX-4T1, was expressed as a fusion protein with GST (GST-mCd x-2D) and purified by one step of affinity chromatography. A polyclonal antibody against Cdx-2 was raised by using the recombinant fusion protein GST-mCdx-2D as antigen and was fractionated from the rabbit anti-serum. Western blot and EMSA (electrophoretic mobility shift assay) demonstrate that the natural and denatured Cdx-2s from different species (mouse and human) can be detected by the prepared anti-Cdx-2 antibody. Most notably, we found that the Cdx-2 in human intestine cell line Caco-2 is expressed in a differentiation-dependent manner and can efficiently bind to the mouse and human acat2 (acyl-coenzyme A: cholesterol acyltransferase 2) promoter regions, suggesting that the transcriptional factor Cdx-2 may play a role in regulating the acat2 expression in the intestinal cells.

Key words Cdx-2; GST fusion protein; polyclonal antibody; EMSA; acat2 promoter

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