NS基因克隆及人成骨肉瘤细胞和人胚胎肾细胞NS基因的表达  

THE CLONING OF NUCLEOSTEMIN GENE FRAGMENT AND THE DETECTION OF ITS EXPRESSION IN OS-732 CELLS AND HEK-293 CELLS

作　　者：刘思金[1] 蔡子微[2] 胡国法[1] 魏影允[1] 劳为德[1] 

机构地区：[1]中国科学院遗传与发育生物学研究所转基因与基因调控研究室,100080 [2]牡丹江医学院病理生理学教研室

出　　处：《牡丹江医学院学报》2003年第2期5-7,共3页Journal of Mudanjiang Medical University

摘　　要：目的 :研究nucleostemin(核干细胞因子 ,NS)基因在人成骨肉瘤细胞 (OS - 732 )和人胚胎肾细胞 (HEK -2 93)的表达情况 ,并克隆该基因的部分片段。方法 :以OS - 732细胞和HEK - 2 93细胞为实验材料 ,进行细胞总RNA的提取、反转录反应、PCR反应、NS基因片段的克隆及测序等。结果 :(1)提取的总RNA质量很高 ,基本上无降解 ;(2 )OS - 732细胞和HEK - 2 93细胞中均有NS基因的表达 ,并且在相同量底物的情况下 ,OS- 732细胞NS基因的表达量明显高于HEK - 2 93细胞 ;(3)将PCR产物成功地与pGEM -T克隆载体连接 ,构建为pGEM -T -NS质粒 ,测序结果表明NS基因片段序列与基因库中登陆的序列完全一致。结论 :本文成功地从OS - 732细胞和HEK - 2 93细胞克隆到NS基因片段 ,OS - 732细胞NS基因的表达量明显高于HEK- 2Objective: To investigate whether the expression of NS gene exists in OS-732 (OS)cells and HEK-293(293) cells, meanwhile to clone the Nucleostemin gene fragment from those cells. Methods:OS cells and 293 cells were cultured in vitro . Such methods as total RNA isolation, gene cloning, and DNA sequencing were used in our research work. Results:(1)We got high quality of total RNA, with almost no degradation.(2)NS gene expresses in both OS cells and 293 cells, moreover the expression level in OS cells was higher than 293 cells. (3)We succeeded in cloning the Nucleostemin gene fragment into pGEM-T vector. The result of DNA sequencing indicated that the NS fragment we cloned was identical to the sequence in Gene Bank. Conclusion:We succeeded in cloning the Nucleostemin gene fragment. The expression in OS cells was holding in a high level, by which it is possible to maintain their self-renewal ability.

关 键 词：核干细胞因子 人成骨肉瘤细胞 NS 基因 克隆 胚胎 肾细胞 反转录反应 PCR反应 

分 类 号：Q344[生物学—遗传学]