Nestin特异性RNA干扰真核表达载体的构建及稳定株筛选  

Construction of human nestin specific short hairpin RNA expressing vector and screening of stable transfected cell clone

作　　者：杨旭辉[1,2] 夏添[2,3] 余伟华[2] 钟跃思[4] 项鹏[2] 何峰[5] 

机构地区：[1]广东省妇幼保健院生殖健康与不孕症科,广东广州510010 [2]中山大学干细胞与组织工程中心,广东广州510080 [3]浙江省台州医院血液肿瘤内科,浙江临海317000 [4]中山大学附属第三医院,广东广州510630 [5]中山大学药学院,广东广州510006

出　　处：《广东药学院学报》2012年第2期204-207,共4页Academic Journal of Guangdong College of Pharmacy

基　　金：国家自然科学基金(30900729;81000177;81173577);广东省自然科学基金(8451008901000380)

摘　　要：目的构建人nestin基因特异的RNA干扰质粒,为探讨抑制nestin基因表达对人黑色素瘤细胞生物学行为调控的研究奠定基础。方法分别用30、50、80 nmol/L干扰或80 nmol/L对照siRNA转染人黑色素瘤细胞株UACC903,48 h后用免疫荧光、RT-PCR和Western-blot鉴定nestin基因表达并筛选有效的序列,用有效或对照组序列的正义链和反义链,通过酶切、连接、转化和筛选,获得长期下调人nestin基因表达的慢病毒载体,并包装慢病毒感染人UACC903细胞,免疫组化和Western-blot鉴定干扰有效,以此获取nestin表达下调的UACC903细胞株。结果重组质粒成功转化高感受态大肠杆菌,经XhoⅠ和HPAⅠ双酶切,琼脂糖凝胶电泳分析,结果表明寡核苷酸成功插入到预计位点,经测序鉴定,序列完全正确。干扰组或对照组慢病毒感染UACC903细胞后与对照组比较,干扰组mRNA和蛋白表达明显降低。结论成功构建了针对人nestin基因的特异性shRNA真核表达载体,转染细胞后可抑制nestin基因的表达,为进一步研究其基因功能奠定了基础。Objective To construct specific human nestin-shRNA expression vector and screen the stable transfected cell clone.Methods Different concentrations of nestin-siRNA（30,50,80 nmol/L） or control siRNA were used to transfect human melanoma cell line UACC903.After 48 hours,nestin expression was detected by immunofluorescence and RT-PCR.The lentivirus vector containing genes of down-regulating nestin expression and two enzyme sites of XhoⅠ-HpaⅠ was constructed and transformed into XL-10 Gold cells.Plasmid was extracted and sequenced to identify effective clone.Recombinant lentivirus was packaged and infected into UACC903 cells.Nestin expression in UACC903 cells was checked.Results Nestin expression in UACC903 was significantly down-regulated by special siRNA treatment as validated by immunofluorence,RT-PCR and Western blotting.Conclusion RNA interfering（RNAi） mediated by the shRNA vector can significantly down-regulate the expression of nestin in UACC903 cells.The stable transfected cell clone is obtained for further study.

关 键 词：RNAI NESTIN 黑色素瘤细胞 

分 类 号：Q786[生物学—分子生物学] R739.5[医药卫生—肿瘤]