Stathmin SiRNA表达载体抑制stathmin的表达

Stathmin SiRNA Plasmid Expression Vector Suppressing Expression of Stathmin

目的:构建stathmin特异性SiRNA质粒表达载体,探讨其对鼻咽癌5-8F细胞stathmin的沉默作用。方法:合成用于stathmin基因特异性干扰表达的DNA片段,经退火形成双链DNA片段,片段克隆到质粒表达载体pGenesil 1.1上。载体导入JM109菌株进行筛选与扩增,采用酶切和测序对克隆表达载体进行鉴定。应用脂质体将鉴定后的重组表达质粒载体转入鼻咽癌5-8F细胞,RT-PCR与Western Blot分析stathmin基因表达。结果:经酶切和测序鉴定,插入SiRNA质粒表达载体的stathmin特异性碱基序列和方向正确。重组质粒表达载体转染鼻咽癌细胞后,细胞转染效率达78.8±6.8%,stathmin基因在鼻咽癌中的表达明显下降。结论:构建的stathmin基因SiRNA质粒表达载体能抑制stathmin的表达。

Objective：Constructing stathmin specific SiRNA plasmid expression vector and investigating the silence effect of SiRNA plasmid expression vector on stathmin in 5-8F cell line.Method：Synthesize stathmin specific interfering expression DNA fragments,obtain doubled DNA fragment by annealing and combine the fragment with plasmid pGenesil1.1.The recombinant vectors were transferred and screened in JM109 strains.Then,the recombinant vectors were amplified and identified by di-enzyme digestion and DNA sequencing.The identified vector was transfected into 5-8F cell line.RT-PCR and Western Blot were adopted to analyze the expression of stathmin.Result：Di-enzyme digestion analysis and DNA sequencing showed that the basic sequence and direction of stathmin specific DNA fragment,which was inserted into SiRNA plasmid expression,was correct and reduced the expression of stathmin effectively in nasopharyngeal carcinoma cell line 5-8F.The transfected efficiency was about 78.8±6.8%.Conclusion：The constructed stathmin specific recombinant SiRNA plasmid expression vector could silence the expression of stathmin.