类表皮生长因子域7基因shRNA慢病毒表达载体的构建及鉴定  

Construction and identification of lentiviral vector-mediated short hairpin RNA of human EGF-like domain 7 gene

作　　者：王爱岳[1] 李强 许琼冠 刘达远 徐鹏翔 余丹[1] 

机构地区：[1]海口市人民医院神经内科,海口570208 [2]海南省农垦总医院神经外科,海口570311

出　　处：《中国临床药理学杂志》2017年第19期1946-1948,共3页The Chinese Journal of Clinical Pharmacology

基　　金：海南省自然科学基金资助项目(814375);海南省卫生厅基金资助项目(琼卫2010-31)

摘　　要：目的用基因重组技术构建LV3-人类EGFL7基因的小发卡RNA(LV3-h EGFL7-shRNA)慢病毒表达载体。方法设计靶向h EGFL7-shRNA序列,建立LV3-h EGFL7-shRNA,在慢病毒载体p GLV3/H1/GFP+Puro中放置h EGFL7-shRNA基因片段,DNA测序以及酶切的方式进行检测h EGFL7片段,转染293T细胞后对上清液进行浓缩,最后在对病毒滴度进行检测。结果 EGFL7-shRNA核苷酸链有正确的插入顺序,包装慢病毒产生病毒悬液的滴度为2×108TU·m L^(-1)。结论 h EGFL7-shRNA慢病毒表达载体构建成功,鉴定可满足试验需求。Objective To construct the LV3-human EGF-like domain 7-targeted small hairpin RNA（ LV3-h EGFL7-shRNA）-expressing plasmid system. Methods Sequence of h EGFL7-targeted shRNA was designed by Oligo Designer 3. 0 based on the mRNA sequence of h EGFL7 which was obtained from the Genbank. They were recombined with the plasmid p GLV3/H1/GFP ＋ Puro,and then those plasmids were identified by gene sequencing to make sure they were correctly connec-ted. Then those plasmids were transfected into 293 T cells and fluorescence expression was observed. Results The sequence of EGFL7-shRNA was demonstrated that the inserted sequences were correct,and the titer of virus was 2×10^8TU·m L^-1 . Conclusion The h EGFL7-targeted shRNA expressing plasmids were successfully constructed,identification can meet test requirements.

关 键 词：类表皮生长因子域7 RNA干扰 短发夹结构RNA 慢病毒 

分 类 号：R97[医药卫生—药品]