Cav2.2e37a基因RNAi慢病毒载体的构建与鉴定

Construction and identification of RNA interference lentivirus vector of Cav2.2 e37a gene

目的构建针对Cav2.2e37a基因的RNAi慢病毒载体,为抑制Cav2.2e37a基因表达治疗神经痛的实验研究打下基础。方法针对已经筛选确定的Cav2.2e37a基因RNAi有效靶序列,构建pLL3.7-Cav2.2e37a干扰质粒,测序鉴定。pRsv-REV,pMDlg-pRRE,pMD2G,pLL3.7-Cav2.2e37a共转染293T细胞,Real-time PCR测定病毒转导滴度。结果成功构建Cav2.2e37a shRNA的慢病毒载体LVshCav2.2e37a。浓缩病毒悬液的滴度为1×109Tu/ml。结论成功构建了Cav2.2e37a基因的RNAi慢病毒载体。

Objective To construct the RNA interference（RNAi） lentivirus vector of Cav2.2 e37a gene in order to inhibit the expression of Cav2.2 e37a gene. Methods An interfere plasmid vector, pLL3.7-Cav2.2e37a, was constructed and confirmed by sequencing according to the effective sequence of siRNA targeting Cav2.2 e37a gene identified in our previous study. 293 T cells were co-transfected with pRsv-REV, pMDlg-pRRE, pMD2G, and pLL3.7-Cav2.2e37a. Concentrated virus titer was detected by realtime PCR. Results The RNAi vector of Cav2.2 e37a gene（pLL3.7-Cav2.2e37a） producing Cav2.2 e37a shRNA was constructed. The concentrated titer of virus suspension was 1×10^9Tu/ml. Conclusion RNAi lentivirus vector of Cav2.2 e37a gene can be constructed.