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目的:构建并筛选针对SD大鼠锌离子转运体1(ZnT1)的siRNA慢病毒载体。并检测其在大鼠神经元中的作用。方法:设计并合成针对ZnT1基因的3条(A,B,C)siRNA序列与1条阴性对照序列,将以上序列退火与pFU-GW-iRNA质粒相连接,通过测序鉴定后,分别与慢病毒包装质粒共感染293T细胞,检测收获病毒滴度后感染体外培养的SD大鼠脑皮层神经细胞,设置感染复数(MOI)分别为1,3,6,8,于转染后72 h计算转染效率。在最佳MOI值下,设置未经病毒感染组(CON组)、阴性对照病毒感染组(NC组)和慢病毒阳性干扰组(ZnT1-siR-NA-A组,ZnT1-siRNA-B组,ZnT1-siRNA-C组),72 h后利用Western blot技术检测ZnT1的表达情况,确定对ZnT1蛋白的最有效的干扰序列与抑制效率。结果:测序证实目的干扰序列已被准确克隆到pFU-GW-iRNA质粒,收获的慢病毒颗粒滴度为8×108TU/ml,其在MOI=6时对神经细胞的转染效率最高(93%),并保持神经细胞良好的生存状态。转染后ZnT1的表达被不同程度的抑制,A,B,C三种干扰序列对ZnT1的抑制率分别为47.74%±1.40%,81.19%±1.36%,94.10%±2.41%。结论:成功构建了ZnT1-siRNA慢病毒载体,其在MOI=6时对神经细胞具有最佳的转染效率,并有效沉默神经细胞中ZnT1蛋白的表达。
OBJECTIVE: To construct and screen siRNA lentiviral vector targeting Zn-Zn transporter 1 in SD rats. And examined its role in rat neurons. Methods: Three (A, B, C) siRNA sequences and one negative control sequence targeting ZnT1 gene were designed and synthesized. The above sequences were annealed to pFU-GW-iRNA plasmid and identified by sequencing. The 293T cells were co-infected with packaging plasmids. The titers of harvested virus were detected and the neurons were cultured in vitro. The multiplicity of infection (MOI) were 1, 3, 6 and 8, respectively. effectiveness. Under the optimal MOI, the mice in the control group (CON group), negative control group (NC group) and lentivirus positive group (ZnT1-siR-NA-A group, ZnT1-siRNA-B group, ZnT1-siRNA-C group). After 72 h, the expression of ZnT1 was detected by Western blot to determine the most effective interference sequence and inhibition efficiency to ZnT1 protein. Results: Sequencing confirmed that the target sequence was correctly cloned into pFU-GW-iRNA plasmid. The titer of the lentiviral particles harvested was 8 × 108TU / ml. The transfection efficiency was the highest (93% ), And maintain a good living condition of nerve cells. The expression of ZnT1 was inhibited to varying degrees after transfection. The inhibitory rates of ZnT1 by A, B and C were 47.74% ± 1.40%, 81.19% ± 1.36% and 94.10% ± 2.41%, respectively. CONCLUSION: The constructed ZnT1-siRNA lentiviral vector has the best transfection efficiency to nerve cells at MOI = 6 and effectively silences the expression of ZnT1 protein in nerve cells.