【摘要】 目的核干细胞因子（nucleostemin,NS）是维持干细胞和癌细胞增殖所必需的蛋白质,可能成为肿瘤基因治疗的潜在靶点.本文旨在构建靶向NS干扰载体p Genesil-1-NS,为后续实验奠定基础.方法基于已发布的NS mRNA序列（NM₂06825）,选取5’-AAGCCTA GGAAAGACCCAGG-3’（397-416）作为候选靶序列,按shRNA载体设计原则,设计合成两条互补DNA链,退火后插入载体,并以酶切和测序鉴定重组转化子.结果经酶切及测序鉴定证实合成序列正确插入载体.结论成功构建NS特异性干扰载体p Genesil-1-NS,可用于NS在肿瘤中的功能研究.更多还原

【Abstract】 Objective Nucleostemin,a protein to maintain the proliferations of stem cell and cancer cell,may become a potential target for tumor gene therapy. This study aimed to construct the NS specific interference vector p Genesil-1-NS,and to lay a foundation for further study. Method Based on the published NS-mRNA sequence（ NM₂06825）,5’-AAGCCTA GGAAAGACCCAGG-3’（ 397-416） was selected as candidate target sequence.According to the shRNA carrier design principle,sh DNA that could be transcripted to shRNA was designed and synthesized,then inserted into p Genesil-1 vector. The recombinant was identified by restriction enzyme digestion analysis followed by sequence analysis. Results Confirmed by restriction analysis and sequencing,sh DNA was correctly inserted into the vector. Conclusion NS-target interference vector p Genesil-1-NS was successfully constructed,which would be applied in the study of NS function in tumor generation and progression.更多还原