CAJ | 학술논문

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。
목적：구건인뇨격매형섬용매원격활인자（uPA）절단형돌변체여록색형광단백（EGFP）분비형융합표체재체병재진핵세포중표체。방법：채용PCR법，분별이질립pIRES2-EGFP화중조질립pcDNA3.1（+）/uPA위모판，확증출대BamHⅠ화XbaⅠ매절위점적EGFP급대NheⅠ화HindⅢ매절위점적uPA절단체기인편단，선후장EGFP화절단형uPA기인편단극륭도진핵표체재체pcDNA3.1（+）상，전입HEK293F세포，용G418대전염세포진행가압사선，통과공취초현미경관찰화ELISA방법감정표체산물。결과：DNA측서결과현시，uPA불동절단형돌변체기인편단여EGFP기인융합적진핵표체재체구건성공，공취초현미경관찰발현HEK293F세포중유록색형광차정위우세포질중，ELISA검측도HEK293F세포배양상청중분비형융합단백적표체。결론：구건료uPA절단형돌변체여EGFP분비형융합표체재체병재진핵세포중표체，위후기연구uPA적상호작용단백급기생리공능전정료기출。
Objective: To construct and express eukaryotic expression vectors of the truncated urokinase-type plas?minogen activator(uPA) fused to enhanced green fluorescent protein(EGFP). Methods: EGFP and truncated uPA genes were amplified by PCR using plasmid pIRES2-EGFP and recombinant plasmid pcDNA3.1(+)/uPA as tem?plates, and inserted into eukaryotic expression vector pcDNA3.1(+) sequentially. The constructed recombinant plas?mids were transfected into HEK293F cells, and treated with high concentration G418. The expression of recombi?nant proteins was detected by confocal microscopy and ELISA. Results: DNA sequencing proved that the eukaryot?ic expression vectors of the fusion proteins were constructed successfully. And the green fluorescent protein could be observed in cells by confocal microscopy after the transfection, and the stable expression cell lines were got af?ter selected by G418. ELISA showed that the secreting type fusion proteins exist in supernatant. Conclusion: Re?combinant plasmids have been constructed and expressed in HEK293T cells, which will contribute to further re?search of the interaction of uPA and its biological function in cells.