下调TPH2重组慢病毒载体的构建及鉴定

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-2 mRNA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-TPH2-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3×108 TU/rnl.结论 获得的Lentivirus-TPH2-siRNA可以用于转基因瘙痒治疗的实验研究.

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

Objective To construct the recombinant lentivirus vector with tryptophan hydroxylase 2-siRNA (TPH 2-siRNA).Methods A target-specific 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 recombinant siRNA vector plasmid pU6-MCS-CMV-TPH2-shRNA.The 293 cells were cotransfected 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 construction of the recombinant Lentivirus-TPH2-siRNA plasmid could be confirmed,and the titer of virus was 3 × 108 TU/ml after purified.Conclusion The Lentivirus-TPH2-siRNA vector can be used in empirical study of transgenic itch therapy.