摘要
目的:构建针对整合素连接激酶(ILK)mRNA的小干扰RNA(siRNA)表达载体,为抑制ILK在哺乳动物细胞内表达、研究其功能奠定基础。方法:合成含靶向ILK基因siRNA转录模板的茎环结构,与两端分别有BamHⅠ、HindⅢ酶切位点的Psilencer 3.1质粒连接,大肠杆菌中扩增,测序鉴定。结果:转化大肠杆菌涂布平板长出阳性菌落,重组质粒电泳初步说明质粒构建成功,测序结果表明Psilencer 3.1酶切位点BamHⅠ、HindⅢ之间有64 bp的插入片段,其序列与所设计、合成的ILK-siRNA转录模板序列一致。结论:siRNA转录模板完整正确地插入Psilencer 3.1质粒中。
Objective To construct ILK-siRNA-DNA expression vector for the further functional research of ILK in mammalian cells by using RNAi. Methods The hairpin structure of ILK-siRNA transcript template was synthesized and then ligated into the linearized Psilencer 3. 1 with T4 DNA ligase. Then E. coli was transformed with the ligation products and the transformed cells were plated on LB plates containing 80 μg · ml^-1 ampicillin. The positive clones were picked and the recombinant plasmid was isolated. Finally,the siRNA template insert was sequenced after plasmid electrophoresis. Results The presence of 64 bp DNA was confirmed after plasmid elelctrophoresis. The insert sequencing showed that the sequence of the insert was identical with what was designed. Conclusion It is confirmed that the recombinant plasmid contains the correct and full ILK-siRNA transcript template.
出处
《东南大学学报（医学版）》
CAS
2006年第6期407-410,共4页
Journal of Southeast University(Medical Science Edition)
基金
国家自然科学基金资助项目(30471732)
关键词
小干扰RNA
质粒
整合素连接激酶
构建
small interfering RNA
plasmid
integrin linked kinase
construction
