[Expression, antibody production and bioactivity detection of cellular repressor of E1A-stimulated gene].
OBJECTIVE
To obtain the human cellular repressor of E1A-stimulated gene (hCREG) protein and the polyclonal antibody against the hCREG, and to further observe the expression and localization of hCREG protein in human internal thoracic artery cells (HITASY).


METHODS
The hCREG cDNA was amplified by PCR and cloned into the pGEX-4T-1. Glutathione-S-transferase (GST)-hCREG fusion protein was expressed in E.coli BL21 and was used to immunize rabbits to obtain anti-hCREG serum, which was purified by protein A and GST immobilized on glutathione-Sepharose beads. The titer and specificity of polyclonal antibodies were determined by ELISA and Western blot analysis. The expression and localization of hCREG protein were detected with immunofluorescence staining in HITASY cells after serum removal. The proliferation of HITASY cells affected by hCREG protein was examined by means of BrdU stain.


RESULTS
It was confirmed that hCREG cDNA was correctly inserted into the vector by endonuclease digesting and DNA sequencing. The expressed GST-hCREG protein was purified by the gel-filtration and its purity was up to about 90%. The anti-hCREG polyclonal antibody was specific with high titer (>1:10(5)) and the hCREG protein was expressed in a perinuclear pattern in HITASY cells after serum deprivation. It was also observed that the proliferation of HITASY cells was obviously inhibited by hCREG protein.


CONCLUSIONS
The hCREG protein was highly expressed in HITASY cells and inhibited HITASY cell proliferation, suggesting that it may be involved in the process of proliferation and differentiation of vascular smooth muscle cells.
Han
Ya-ling
YL
Cardiovascular Institute of PLA, Department of Cardiology, General Hospital of Shenyang Command of Chinese PLA, Shenyang 110016, China. hanyal@mail.sy.ln.cn
Liu
Hai-wei
HW
Yan
Cheng-hui
CH
Kang
Jian
J
Wang
Xiao-zeng
XZ
Hu
Ye
Y
D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D008323:Mammary Arteries / Q000166:cytology; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*
C114068:CREG1 protein, human; D012097:Repressor Proteins