PTHrP基因靶向RNA干扰重组表达质粒的构建鉴定以及在HTB-94细胞中的功能检测  

Construction and Identification of SiRNA Expression Plasmid Targeting PTHrP and Function in Human Chondrosarcoma Cells

作　　者：秦宏敏[1] 韩会峰[1] 沙广钊 

机构地区：[1]徐州医学院附属医院创伤骨科,江苏省徐州市221002 [2]江苏邳州第一人民医院

出　　处：《中国骨与关节损伤杂志》2008年第7期564-566,共3页Chinese Journal of Bone and Joint Injury

基　　金：江苏省自然科学基金(05KJB320135);江苏省高校自然科学研究基金资助项目(BK2005015)

摘　　要：目的构建针对目的基因PTHrp的pSilencer3.1-H1neo siRNA(small interfering RNA)表达质粒,并在人体软骨肉瘤细胞HTB-94中观察其干扰效果。方法设计并合成针对PTHrP基因编码区的含有发卡结构的互补的寡核苷酸链,体外退火后克隆入经BamHⅠ,HindⅢ双酶切线性化的干扰载体pSilencerTM3.1H1neo中,对重组质粒进行酶切分析和DNA系列测定。结果双酶切证实PTHrP siRNA表达质粒克隆构建成功,插入片段测序结果与合成的siRNA结果一致,同时干扰了PTHrP基因的mRNA和蛋白的表达。结论成功构建的靶向RNA干扰重组表达质粒,可以通过小RNA序列干扰靶向基因的mRNA转录,降低靶向基因在细胞中mRNA和蛋白的表达。Objective To construct pSilencerTM 3.1H1 small inferfering RNA （siRNA） expression plasmid targeting human PTHrP gene and to observe its silencing effect in the human HTB - 94 chondrosarcoma cells. Methods Two DNA sequences containing small hairpin structure were designed and synthesized. The designed oligos targeting PTHrP were cloned into the pSilencerTM 3.1H1 neo vector, which was lineated after BamH I and Hind Ⅲ digestion. The recombinant plasmid was confirmed by enzyme digestion analysis and DNA sequencing. Results siRNA （PTHrP） expression plasmid was successfully constructed and identified by double endonuclease digestion. Sequence analysis of the inserted fragment revealed the same sequence as that of the synthesized siRNA oligonucleotides. Meanwhile the mRNA and protein expression of PTHrP in HTB- 94 cells which was interfered by siRNA （PTHrp） plasmid were significantly reduced. Conclusion siRNA （PTHrP） expression plasmid could interfere with the target gene RNA transferring through small inferfering RNA, and reduce the mRNA and protein expression of the target cell gene.

关 键 词：siRNA(PTHrp)质粒构建 RNA干扰 PTHrp蛋白和mRNA表达 

分 类 号：R378.99[医药卫生—病原生物学] R394[医药卫生—基础医学]