Polyclonal antibody production and expression of CREG protein in human vascular smooth muscle cells

Objectives The cellular repressor of El A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E.coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot analysis detected the expression of smooth muscle-specific markers (SM a-actin, Calponin). The localization of CREG protein was examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG protein, SM a-actin and Calponin is induced respectively in HITASY cells after serum deprivation. Conclusions The specificity of polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.(J Geriatr Cardiol 2005,2(2): 118-122).