Establishment of 5-lipoxygenase inducible expression system / 第二军医大学学报

ABSTRACT

To develope a 5 lipoxygenase inducible expression system for further understanding the function and signal transduction pathway of 5 lipoxygenase, providing information for drug screening.The 2 kb 5 LO cDNA sequence containing HindⅢ site was generated by PCR using pEGFP 5LO as a template. Full length 5LO cDNA was subsequently cloned into the corresponding Pst Ⅰ and Sal Ⅰ sites of the plasmid pBI G to generate the mammalian expression vector pBI 5LO. HeLa Tet On cell line was co transfected with pBI 5LO and pTK Hyg plasmids. Inducible expression of target gene was detected using X gal histochemical staining of cell monolayers for ? galactosidase after incubating the transfected cell line with 1 ?g/ml doxycycline for 48 h.The HindⅢ restriction digest site was inserted into polyclonal site of pBI G and recombinant vector pBI 5LO was constructed. The desired recombinant plasmid pBI 5LO was identified by restriction analysis, and orientation and junctions were confirmed by sequencing. Induction of the target gene expression by doxcycline treatment was demonstrated by X gal histochemical staining and nuclei of the cells that have expressed the pBI 5LO expression vector were brilliant blue.The 5LO inducible expression system is established successfully and it works well in cell model.