基于Te-OnAdvanced的肺癌细胞15-脂氧化酶-2基因表达载体的构建与表达

目的：构建肺癌细胞15-脂氧化酶-2（15-Lox-2）的可诱导性真核表达载体pTRE-Tight-15-Lox-2，并在肺癌细胞中检测其是否可受强力霉素（DOX）诱导表达。方法：pcDNA3-15-LOX-2载体经＆0RI和XbaI双酶切线性化，回收15-LOX-2cDNA片段，将其克隆入pTRE-Tight载体的EcoRI和XbaI位点；采用脂质体法将pTet-0n-Ad-vanced与构建的pTRE-Tight-15-Lox-2共转染肺腺癌A549细胞，DOX诱导表达后，Western印迹检测15-Lox-2的表达水平。结果：构建了pTRE-Tight-15-Lox-2诱导表达载体；Western印迹检测表明，该载体能在肺癌细胞内表达，且其表达受DOX调控。结论：Tet-OnAdvanced系统能严密高效地调控15-LOX-2在肺癌细胞中的表达，为进一步研究15-LOX-2在肺癌中的作用奠定了基础。

Construction and Expression of 15-Lipoxygenase-2 Gene Vector Regulated by Tet-On Advanced System in Lung Cancer Cells

Objective： To construct tetracycline （Tet） -controlled inducible vector pTRE-Tight-15-LOX-2, and to determinate expression character of 15-1ipoxygenase-2（15-LOX-2） under the regulation of doxycline （DOX） in lung cancer ceils. Methods： pcDNA3-IS-LOX-2 vector was linearized with EcoR I and Xba I restriction endonu- cleases and 15-LOX-2 cDNA was retrieved and cloned into the EcoR I and Xba I sites of pTRE-Tight vector. pTet-On-Advanced and pTRE-Tight-15-LOX-2 were cotransfected into A549 lung cancer cells using Lipo- fectAMINE 2000. Expression of 15-LOX-2 was assayed by Western blot after giving DOX. Results： Inducible ex- pression vector pTRE-Tight-15-LOX-2 was successfully constructed. Western blot showed that expression of 15- LOX-2 could be induced by DOX in lung cancer cells. Conclusion： The Tet-On Advanced system can tightly and robustly regulate the expression of 15-LOX-2 in lung cancer cells, and this work has laid foundations for fur- ther study on 15-LOX-2 in lung cancer.