大鼠Rars基因siRNA重组腺病毒载体的构建与鉴定

目的 构建针对大鼠Rars基因的siRNA重组腺病毒载体，并观察大鼠神经元细胞病毒转染前后目的蛋白表达量变化。方法 将原代培养的大鼠神经元细胞随机分为观察组、阴性对照组及空白对照组。观察组细胞转染携带Rars siRNA序列的重组腺病毒，阴性对照组细胞转染携带Scramble无意义序列的重组腺病毒，空白对照组不转染任何病毒。观察各对照组表达绿色荧光蛋白（GFP）的阳性细胞数，计算转染率，并检测转染前后Rars基因对应产物精氨酰-tRNA合成酶（ArgRS）蛋白的表达量，评价基因沉默效率。结果 成功构建包含Rars基因干扰序列及Scramble序列的重组腺病毒，病毒滴度达1010级。利用重组腺病毒感染大鼠神经元细胞48 h后，绝大部分细胞表达GFP，转染率>95%。含Rars干扰序列的病毒转染细胞可使ArgRS蛋白表达量显著降低（P<0.01），沉默效率>60%。含Scramble对照序列的病毒则不能产生明显的基因沉默效应。结论 本实验所构建的siRNA重组腺病毒载体可有效沉默大鼠Rars基因，降低ArgRS蛋白表达。

Construction and evaluation of rat Rars siRNA recombinant adenoviral vector

Objectives To construct the recombinant adenoviral vector carrying rat Rars small interfering RNA (siRNA) gene and to investigate changes in the ArgRS protein (the translation product of Rars) level after the viral transfection in rat neurons. Methods The neurons derived from SD rat brain which were primarily cultured were randomly divided into three groups, i.e. experimental group in which the neuron were transfected with recombinant viral vectors carrying Rars siRNA sequence, negative control group in which the neurons were trhansfected with vectors carrying Scramble sequence (nonsense sequence) and the blank control group. The transfection rate was calculated by counting green fluorescence protein (GFP) positive cells 48h after the transfection. The inhibition rate of Rars gene was evaluated by observing the ArgRS expressions levels before and after the virus transfection. Results The recombinant adenoviral vectors carrying Rars siRNA sequence and Scramble sequence were successfully constructed and the tilter of the virus was 1010 pfu/ml. The most cells expressed GFP and the transfection rate was above 95% 48 hours after transfection. The levels of ArgRS expression in the neurons was significantly lower in the experimental group in which the inhibition rate was over 60% than those in both the control groups (P<0.01). Conclusions Rars siRNA recombinant adenoviral vector constructed in the present study can effectively silence Rars gene and downregulate ArgRS expression in the neurons of the rats.