摘要
目的利用RNA干扰技术,以Akt2为靶基因,设计并构建重组表达载体,并进行测序鉴定。方法利用GenBank中已知Akt2的mRNA基因序列设计具有短发夹结构的两条寡核甘酸序列,经退火形成互补双链,与pGEM-T Easy载体进行连接,经蓝白筛选后以T7和SP6为引物进行PCR鉴定,对表达载体pRNAT-U6.2及亚克隆产物进行双酶切,将得到的基因及线性化的表达载体再次连接,利用PCR方法进行重组体的筛选鉴定并进行测序分析。结果将合成的DNA序列克隆到表达载体上,经PCR扩增筛选鉴定和测序鉴定证实为所需序列。结论Akt2靶向RNA干扰重组表达载体构建成功。
Objective To construct Akt2 siRNA eukaryotic expression vectors and identify it＇s sequence. Methods Using the known genic sequence of Akt2 mRNA, compose two oligonucleotides that have short hairpin configuration, anneal to double chain,connect this constructed genic segment with clone vector pGEM-T Easy, blue-white selection test and T7/ SP6 PCR were used to screen the positive clones, then cut down the expression vector pRNAT-U6.2 and the outcome of sub-clone by BamHI and XhoI restriction enzyme and connect the target DNA with the lined expression vector, use PCR test to filtrate the positive clones and sequenced the sequence . Results Targeting sequences of Akt2 siRNA eukaryotic expression vectors were correct. Conclusion Two Akt2 siRNA eukaryotic expression vectors were constructed successfully.
出处
《山东医药》
CAS
北大核心
2008年第28期19-21,共3页
Shandong Medical Journal
基金
河南省卫生厅科技攻关课题(20060035)
