Preparation
of an Anti-Cdx-2 Antibody for Analysis of Different Species Cdx-2 Binding to acat2
Promoter
SONG Bao-Liang1, QI Wei1,2, WANG Can-Hua1,3,
YANG Jin-Bo1, YANG Xin-Ying1, LIN Zhi-Xin3, LI
Bo-Liang1*
( 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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