5-脂氧合酶诱导表达系统的建立  

Establishment of 5-lipoxygenase inducible expression system

作　　者：周斌[1] 陈新生[1] 殷明[1] 

机构地区：[1]第二军医大学药学院药理学教研室,上海200433

出　　处：《第二军医大学学报》2003年第2期225-227,共3页Academic Journal of Second Military Medical University

基　　金：国家杰出青年科学基金资助项目 (3 992 5 0 3 8)

摘　　要：为建立 5 -脂氧合酶 (5 L O)诱导表达系统 ,深入研究 5 L O功能、信号转导机制及药物筛选 ,采用 PCR扩增含 H ind 酶切位点的 2 kb长的 5 L O c DNA基因片段并将其克隆入质粒 p BI- G中构建重组表达载体 p BI- 5 L O。 p BI- 5 L O/ p TK- Hyg共转染 Tet- He L a细胞。多西环素与转染细胞孵育 4 8h诱导目的基因表达。对构建的重组载体 p BI- 5 L O进行序列测定 ,证实克隆的目的基因片段是 5 L O基因片段 ,且阅读框架正确。X- gal染色证实多西环素能诱导目的基因表达 ,阳性细胞的细胞核被染为蓝色。提示 5 L O诱导表达系统建立成功 。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 β galactosidase after incubating the transfected cell line with 1 μg/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.

关 键 词：5-脂氧合酶 诱导表达 信号转导 药物筛选 

分 类 号：R96[医药卫生—药理学]