CAJ | 학술논문

目的：构建携带多瘤病毒MT癌基因的真核细胞表达载体。方法：从野生型多瘤病毒上取其nt4632至nt1560片段克隆到pUC19上，再将该片段定点插入pEGFP1的HindⅢ和EcoRI位点间，构建携带多瘤病毒MT癌基因的真核表达载体pPyMT-GFP。经线性化，将重组体导入鼠皮肤成纤维细胞中，G418选择培养，得到抗性细胞克隆。结果：构建的pPyMT-GFP质粒在鼠成纤维细胞中有稳定表达。结论：来源于野生型多瘤病毒的PyMT基因在哺乳动物细胞中有稳定表达，其真核表达载体pPyMT-GFP可被进一步用于研究PyMT蛋白在血管瘤发生中的作用。
목적：구건휴대다류병독MT암기인적진핵세포표체재체。방법：종야생형다류병독상취기nt4632지nt1560편단극륭도pUC19상，재장해편단정점삽입pEGFP1적HindⅢ화EcoRI위점간，구건휴대다류병독MT암기인적진핵표체재체pPyMT-GFP。경선성화，장중조체도입서피부성섬유세포중，G418선택배양，득도항성세포극륭。결과：구건적pPyMT-GFP질립재서성섬유세포중유은정표체。결론：래원우야생형다류병독적PyMT기인재포유동물세포중유은정표체，기진핵표체재체pPyMT-GFP가피진일보용우연구PyMT단백재혈관류발생중적작용。
Objective:To construct a plasmid for exploring the role ofpolyoma virus middle T (PyMT) protein in the development of hemangioma. Methods:The open reading frame of PyMT gene from Polyoma virus (Py) genome deleted replication starting sites was digested with restricted enzyme and subcloned into pUC19 plasmid.The resulting plasmid pPyMT was digested with HindⅢ and EcoRI.The PyMT fragment was inserted to plasmid pEGFP1. The recombinant plasmids with proper orientation were identified with analysis of restriction enzymes and PCR.The recombinant vector pPyMT-GFP and the vector-alone pEGFP1 were lined and transfected into mouse skin fibroblasts with electroporation technique.After selected with G418, resistant colonies were obtained. Results:The results showed that the recombinant plasmid could express PyMT efficiently under the control of polyoma virus promoter and enhancer. Conclusions:Because the eukaryotic expression vector pPyMT is derived from the polyoma virus, the PyMT gene could express steadily in mammalian cells.The recombinant vector pPyMT-GFP could be used further to study the effect of PyMT protein on the development of hemangioma.