Construction of decorin expression vector and its expression in CHO cells / 吉林大学学报(医学版)

ABSTRACT

Objective To construct a eukaryotic expression vector of decorin(DCN),and observe its expression in CHO cells,in order to provide a basis for further study on the anti-tumor effect of DCN. Methods DCN cDNA was amplified by PCR.The human full-length DCN cDNA ligated into pBluescript was used as template.The fragment was ligated to the expression vector pCDNA3 previously digested with XbaⅠ and EcoR Ⅰ.The ligation mixture was transformed into competent E.coli JM109 cells.Transformants containing inserts were confirmed by restrictive digestion and DNA sequencing.The expression vector was transfected into CHO cells using lipofectamine,and transfected cells were cultivated in DMEM containing G418 (800 mg?L-1) for about 2 months.Immunohistochemistry method was used to detect the expression of DCN protein in stably transfected cells.Results The PCR product was about 1 000 bp.The recombinant expression vector was identified by restrictive digestion and DNA sequencing. DCN protein was detectable in stablely transfected cells.Conclusion The recombinant eukaryotic expression vector pCDNA-DEC is constructed successfully and stablely transfected CHO cells are established.