人尿激酶型纤溶酶原激活因子截短型突变体与绿色荧光蛋白在真核细胞HEK293F中的融合表达  

Expression of Truncated uPA Fused to EGFP in HEK293F Cells

作　　者：张连成[1] 高丽华[1] 张昕[2] 潘芸[2] 高招刚[2] 李伊培[3] 胡显文[1] 陈惠鹏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]安徽大学生命科学学院,安徽合肥230601 [3]沈阳药科大学生命科学学院,辽宁沈阳110016

出　　处：《生物技术通讯》2013年第3期320-324,共5页Letters in Biotechnology

基　　金：国家自然科学基金(81202445;30973670);重大新药创制国家科技重大专项(2011ZX09102-001-30;2012ZX09102301-001)

摘　　要：目的:构建人尿激酶型纤溶酶原激活因子(uPA)截短型突变体与绿色荧光蛋白(EGFP)分泌型融合表达载体并在真核细胞中表达。方法:采用PCR法,分别以质粒pIRES2-EGFP和重组质粒pcDNA3.1(+)/uPA为模板,扩增出带BamHⅠ和XbaⅠ酶切位点的EGFP及带NheⅠ和HindⅢ酶切位点的uPA截短体基因片段,先后将EGFP和截短型uPA基因片段克隆到真核表达载体pcDNA3.1(+)上,转入HEK293F细胞,用G418对转染细胞进行加压筛选,通过共聚焦显微镜观察和ELISA方法鉴定表达产物。结果:DNA测序结果显示,uPA不同截短型突变体基因片段与EGFP基因融合的真核表达载体构建成功,共聚焦显微镜观察发现HEK293F细胞中有绿色荧光且定位于细胞质中,ELISA检测到HEK293F细胞培养上清中分泌型融合蛋白的表达。结论:构建了uPA截短型突变体与EGFP分泌型融合表达载体并在真核细胞中表达,为后期研究uPA的相互作用蛋白及其生理功能奠定了基础。Objective： To construct and express eukaryotic expression vectors of the truncated urokinasetype plasminogen activator（uPA） fused to enhanced green fluorescent protein（EGFP）. Methods： EGFP and truncated uPA genes were amplified by PCR using plasmid plRES2-EGFP and recombinant plasmid peDNA3.1（＋）/uPA as templates, and inserted into eukaryotie expression vector peDNA3.1（＋） sequentially. The constructed recombinant plasraids were transfected into HEK293F cells, and treated with high concentration G418. The expression of reeombinaut proteins was detected by confocal microscopy and ELISA. Results： DNA sequencing proved that the eukaryotic expression vectors of the fusion proteins were construeted successfully. And the green fluorescent protein could be observed in ceils by confoeal microscopy after the transfection, and the stable expression cell lines were got after selected by G418. ELISA showed that the secreting type fusion proteins exist in supernatant. Conclusion： Recombinant plasmids have been constructed and expressed in HEK293T cells, which will contribute to further research of the interaction of uPA anti its biological function in cells.

关 键 词：尿激酶型纤溶酶原激活因子 绿色荧光蛋白 真核表达 融合蛋白 

分 类 号：Q78[生物学—分子生物学]