可调控小鼠尿激酶原激活剂真核表达载体的构建及其在Huh7细胞中的表达

Constrution of Tetracycline -Inducible Mice Urokinase Plasminogen Activator Eukaryotic Expression Vector and its Expression in Huh7 Cells

目的:建立基于Tet-on系统的可调控真核细胞表达小鼠尿激酶原激活剂(uPA)的诱导表达系统。方法:提取C57小鼠肾组织总RNA,RT-PCR扩增uPAcDNA序列;提取基因组DNA,扩增uPA编码区后最后一个外显子序列,构建pTRE2-uPAcDNA-700载体,将其与pTet-on瞬时共转染Huh7细胞系,24h后用强力霉素诱导表达,诱导后36h分别收集细胞和培养上清(诱导组),提取细胞总RNA并进行细胞uPA转录水平的检测;采用溶圈法对细胞分泌至培养上清中uPA的生物活性进行检测,同时以转染但未诱导Hun7(未诱导组)和正常培养Huh7(正常对照组)细胞及培养上清作为对照。结果:与未诱导组和正常对照组相比较,仅诱导组在转录水平上扩增出目的条带;溶圈法证实转染的细胞不仅表达uPA,而且表达的蛋白具有一定的生物活性。结论:构建了可调控的uPA真核诱导表达系统,为进一步制备可调控肝细胞表达uPA转基因小鼠及进一步揭示uPA肝损伤机理奠定了基础。

Objective： To construct a tetracycline-inducible eukaryotie expression system of mice urokinase plasminogen activator（uPA）. Methods： The total RNA and DNA were extracted from normal mice kidney. Mice uPA cDNA fragment and exon 11 was subcloned by RT-PCR and PCR respectively, and pTRE2-uPAcDNA-700 was constructed by inserting the cDNA fragment and exon 11 into the restriction site of the inducible eukaryotic expression vector pTRE2. The Huh7 cells cotransfected by pTet-on and pTRE2-uPAcDNA-700 was conducted followed by doxycycline （Dox） 24 hours later to induce the expression of uPA. Another 36 hours later, the cells and supernatant（Dox group） were collected respectively and the expression and biological activity of uPA were detected. The cells and supematant of cotransfection without Dox-induced as non-inducible group, and that of nomal Huh7 cells as control group. Results： Compared with controlgroup and non-inducible group, RNA extracted from cells of inducible group amplified the interesting band, the expression and biological activity of uPA could be detected. Condusion： The tetracycline inducible eukaryotic expression system of mice u- PA was constructed successfully, which set good basis for the further study of inducible liver-specific uPA expression mice model and the mechanism of liver damage caused by the expression of uPA.