摘要
目的构建靶向Survivin基因及CDK1基因的短发夹样RNA(Short hairpin RNA,shRNA)真核表达载体。方法根据Genbank报道的Survivin序列及CDK1序列,遵循shRNA设计原则设计并合成各自靶向的Survivin、CDK1基因的shRNA寡核苷酸序列,构建pU6-shRNA-Survivin重组质粒、pU6-shRNA-CDK1重组质粒及pU6-shRNA-Survivin-U6-shRNA-CDK1双基因序列串联重组质粒,并进行限制性内切酶酶切及基因测序鉴定。结果经酶切及测序结果分析,pU6-shRNA-Survivin重组质粒、pU6-shRNA-CDK1重组质粒及pU6-shRNA-Survivin-U6-shRNA-CDK1双基因系列串联重组质粒均成功构建。结论成功构建Survivin基因及CDK1基因联合靶向shRNA重组质粒,为进一步研究Survivin基因和CDK1基因联合干扰提供了新的方法。
Objective To construct recombinant survivin and CDK1 tandem shRNAs pU6-M4 plasmids. Methods Sequences of survivin and CDK1 and the restriction enzyme cutting sites of them were designed and synthesized based on the date from GeneBank. Recombinant plasmids of pU6-M4-Survivin-shRNA and pU6-M4-CDKI-shRNA were constructed and identified. Two determined plasmids were digested by restriction endonucleases then taped by T4 DNA ligase. The ligated products were transformed into competent THSa ceils, and then the recombinant clones were identified by sequencing. Results Restriction enzyme cleave identification and sequencing proved that recombinant survivin and CDK1 tandem shRNAs pU6-M4 plasmids were successfully constructed. Conclusion Recombinant survivin and CDK1 tandem shRNAs pU6-M4 plasmids were correctly constructed.
出处
《海南医学》
CAS
2014年第13期1873-1877,共5页
Hainan Medical Journal
基金
国家自然科学基金资助项目(编号:81060223)
