摘要
目的构建Cdc42-shRNA真核表达载体。方法合成Cdc42的基因干扰序列并定向插入到RNA干扰(RNAi)真核表达载体pGenesil-1质粒,通过测序进行鉴定。干扰质粒经脂质体Lipofectamine2000介导转染肝癌细胞,Westernblot方法检测沉默效率。结果测序证明干扰序列完全正确,Westernblot结果显示Cdc42-shRNA对肝癌细胞株内源性Cdc42有较好的沉默效果。结论成功构建了Cdc42-shRNA真核表达载体,为进一步研究Cdc42在基因治疗中的作用奠定基础。
Objective To construct the Cdc42-shRNA eukaryotic expression vectors. Methods Cdc42 gene interference sequence was synthesized and inserted into pGenesil-1 vector, which was confirmed by sequencing. The recombinant RNAi vectors were tmnsfected into hepatic carcinoma cells by Lipofeetamine 2000. Westemblot was then used to investigate the interfering efficiency. Restills Sequencing sugested that RNAi eukaryotie expression vectors targeting Cdc42 possessed correct nucleotide sequence. Western-blot revealed that Cdc42-shRNA could efficiently suppress Cdc42 expression in hepatic earcionma cells. Conclusion Cdc42 shRNA eukaryotie expression vectors were successfully constructed. This work has laid a solid foundation for further study on cancer gene therapy.
出处
《中国实验诊断学》
北大核心
2010年第4期505-507,共3页
Chinese Journal of Laboratory Diagnosis
