下调脂肪酰胺水解酶重组慢病毒载体的构建和鉴定  

Construction of recombinant lentivirus vector with fatty acid amide hydrolase-short hairpin RNA

作　　者：丁得方[1] 李荣春[1] 周伶[1] 

机构地区：[1]华中科技大学同济医学院附属普爱医院疼痛科,武汉430010

出　　处：《中华实验外科杂志》2014年第4期838-839,共2页Chinese Journal of Experimental Surgery

基　　金：湖北省卫生厅科研项目（JX6835）;武汉市卫计委科研项目（WX13815）

摘　　要：目的构建携带脂肪酰胺水解酶（FAAH）基因的小分子干扰RNA表达载体。方法设计合成短发卡RNA（shRNA）FAAH对应的两条互补的寡核苷酸链，mU6-MCS-Ubi-EGFP质粒经HpaI和XhoI进行双酶切与退火后的寡核苷酸连接，目的质粒转化感受态细胞，对克隆的菌落行聚合酶链反应鉴定，再对聚合酶链反应（PCR）鉴定阳性的克隆进行测序，测序正确的重组质粒转染293细胞，同源重组产生Lentivirus-FAAH-shRNA并测定病毒滴度。结果经测序证实mU6-FAAshRNA-Ubi-EGFP表达载体构建正确，并且病毒滴度为7．0×10^8TU/ml。结论实验成果构建mU6-FAAH-shRNA-Ubi-EGFP表达载体。Objective To construct the recombinant lentivirus vector with fatty acid amide hydro lase （FAAH）-short hairpin RNA （shRNA）. Methods Oligonucleotide containing the small hairpin of FAAH was designed and synthesized, which was inserted into the mU6-MCS-Ubi-EGFP plasmid double di- gested by Hpa I and Xho I. The aim plasmids were transformed into competent cells E. coll. The grown colonies were identified by polymerase chain reaction （PCR） and then the positive colonies were sequenced and aligned. The 293 cells were cotransfected by the mU6-FAAH-shRNA-Ubi-EGFP, and the recombinant vector of Lentivirus-FAAH-shRNA was obtained and the titer of purified virus was determined. Results The gene sequencing showeded that the construction of the recombinant Lentivirus-FAAH-shRNA plasmid could be confirmed, and the titer of virus was 7.0 × 10^8 TU/ml. Conclusion The Lentivirus-FAAH-shRNA had been constructed successfully.

关 键 词：脂肪酰胺水解酶 重组质粒 RNA干扰 

分 类 号：R346[医药卫生—基础医学]