携带VGLUT3-shRNA重组慢病毒的制备和鉴定  

Construction of recombinant lentivirus vector with vesicular glutamate transporter 3-short hairpin RNA

作　　者：张红莉[1] 郭晴睛[2] 周伶[2] 丁得方[2] 周外平[2] 冯觉平[3] 李荣春[2] 

机构地区：[1]深圳市宝安区松岗人民医院急诊科,518105 [2]华中科技大学同济医学院附属普爱医院疼痛科 [3]华中科技大学同济医学院附属普爱医院肿瘤科

出　　处：《中华实验外科杂志》2012年第7期1377-1378,共2页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81071307）;华中科技大学自主创新研究基金前沿探索项目（2012TS060）;中央高校基本科研业务费专项资金资助项目

摘　　要：目的制备携带谷氨酸转运体3（VGLUT3）基因小分子干扰RNA的重组慢病毒。方法设计合成短发卡RNA（shRNA）VGLUT3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（PCR）鉴定，再对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—VGLUT3-shRNA并测定病毒滴度。结果病毒滴度为1．5×10^9TU／ml。结论成功制备携带VGLUT3-shRNA的重组慢病毒。Objective To construct recombinant lentivirus with vesicular glutamate transporter 3 （VGLUT3）-short hairpin RNA （shRNA）. Methods Oligonucleotide containing the small hairpin of VGLUT3 was designed and synthesized, which was inserted into the mU6-MCS-Ubi-EGFP plasmid double digested by Hpa I and Xho I. The aim plasmids were transformed into competent cells E. coll. The grown colonies were identified by colony polymerase chain reaction （PCR） and then the positive colonies were se- quenced and aligned. The 293 cells were cotransfected by the mU6-VGLUT3-shRNA-Ubi-EGFP, and the recombinant vector of Lentivirus-VGLUT3-shRNA was obtained and the titer of purified virus was deter- mined. Results The titer of virus was 1.5 × 109 TU/ml. Conclusion The Lentivirus-VGLUT3-shRNA had been constructed successfully.

关 键 词：谷氨酸转运体3 重组慢病毒 RNA干扰 

分 类 号：Q[生物学] R[医药卫生]