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目的:构建Nucleostemin(NS)基因真核表达载体,并在COS-7细胞中表达。方法:设计引物在肺癌细胞株(LTEP-a-2)中扩增NS全长cDNA片段,插入pMD-18T质粒中得pMD-18T-NS载体,并测序;再将pMD-18T-NS质粒经酶切后导入真核表达载体pcDNA3.1(+)-GFP质粒中,构建pcDNA3.1(+)-GFP-NS载体,并检测其真核表达情况。结果:经RT-PCR扩增得到1650bp的全长NScDNA片段,酶切及测序后鉴定证明pMD-18T-NS构建成功。pcDNA3.1(+)-G-FP-NS载体转染COS-7细胞经蛋白印迹及激光共聚焦显微镜检测显示,该GFP-N-S融合基因在COS-7细胞中获得较好表达。转染后见该基因定位于核仁,细胞逐渐失去黏附贴壁的特性,细胞的增殖受阻;稳定表达(4～20周)后,细胞形态发生改变,体积增大,核增多,细胞成瘤细胞样改变。结论:成功构建pcDNA3.1(+)-GF-P-NS真核表达载体,并在COS-7细胞中有效表达;NS基因在COS-7发生瘤样改变过程中发挥着重要作用,可能与肿瘤发生密切相关。
Objective: To construct eukaryotic expression vector of Nucleostemin (NS) gene and express in COS-7 cells. Methods: Primer was designed to amplify NS full-length cDNA fragment in lung cancer cell line (LTEP-a-2), and inserted into pMD-18T plasmid to obtain pMD-18T-NS vector and sequenced. Plasmid pMD-18T-NS The recombinant plasmid pcDNA3.1 (+) - GFP-NS was subcloned into the eukaryotic expression vector pcDNA3.1 (+) - GFP to construct eukaryotic expression vector pcDNA3.1 (+) - GFP-NS. Results: The 1650bp full length NScDNA fragment was amplified by RT-PCR. After digestion and sequencing, pMD-18T-NS was successfully constructed. Transfection of COS-7 cells with pcDNA3.1 (+) - G-FP-NS vector by Western blotting and confocal laser scanning microscopy showed that the GFP-N-S fusion gene was well expressed in COS-7 cells. After transfection, the gene was located in the nucleolus, and the cells gradually lost the characteristics of adhering to the adherent wall. The proliferation of the cells was blocked. After stable expression (4 to 20 weeks), the cell morphology changed, the volume increased, the nucleus increased, Cell-like changes. CONCLUSION: The eukaryotic expression vector pcDNA3.1 (+) - GF-P-NS was successfully constructed and efficiently expressed in COS-7 cells. The NS gene plays an important role in tumor-like changes of COS-7, Tumor is closely related.