下调TPH2重组慢病毒载体的构建及鉴定  

Construction of the recombinant lentivirus vector with tryptophan hydroxylase 2-siRNA

作　　者：戴少军 杨少兵[3] 刘成[3] 李荣春[2] 项红兵[3] 

机构地区：[1]武汉市商业职工医院麻醉科,430021 [2]武汉市普爱医院疼痛科 [3]华中科技大学同济医学院附属同济医院麻醉科

出　　处：《中华实验外科杂志》2012年第1期129-131,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81071307）

摘　　要：目的构建色氨酸羟化酶（TPH）-2基因小干涉RNA的重组慢病毒载体。方法在TPH-2mRNA序列中选择1个特异性靶序列，体外合成对应发卡样DNA片段，经退火后，将其定向克隆入siRNA载体，获得重组质粒pU6-MCS—CMV—rrPH2-shRNA，后者与慢病毒试剂共转染293细胞，同源重组产生Lentivirus—TPH2-siRNA。经聚合酶链反应（PCR）鉴定目的基因的表达并测定病毒滴度。结果PCR表明Lentivirus—TPH2-siRNA构建正确，病毒滴度为3×10^8TU／ml。结论获得的Lentivirus—TPH2-siRNA可以用于转基因瘙痒治疗的实验研究。Objective To eonstmet the recombinant lentivirus vector with tryptophan hydroxylase 2-siRNA （TPH 2-siRNA）. Methods A target-speeific sequence from TPH2 mRNA was used to synthesize the corresponding hairpin DNA fragments in vitro. After annealing, the DNA products were cloned into siRNA vector to obtain the reeombinant siRNA vector plasmid pU6-MCS-CMV-TPH2-shRNA. The 293 cells were eotransfeeted by the Lentivirus vector and pU6-MCS-CMV-TPH2-shRNA, and the recombinant vector of Lentivirus-TPH2-siRNA was obtained. The expression of the transfected genes was evaluated by polymerase chain reaction （PCR） and the titer of purified virus was determined. Results The identification of PCR showed that the eonstnmtion of the recombinant Lentivirus-TPH2-siRNA plasmid eould be con-firmed, and the titer of virus was 3×10^8 TU/ml after purified. Conclusion The Lentivirus-TPH2-siRNA veetor ean be used in empirieal study of transgenic iteh therapy.

关 键 词：色氨酸羟化酶 小干涉RNA 慢病毒 遗传载体 

分 类 号：R758[医药卫生—皮肤病学与性病学]