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目的:拟采用RNA干扰技术筛选有效的针对LCRG1基因的打靶序列。方法:首先采用PCR定点突变技术改造pSuper载体,而后利用该改造后的载体构建针对LCRG1基因的5对打靶序列的真核表达载体。将构建的重组pSuper362,398,432,789,903表达载体和pSuper空白载体分别转染He-la细胞,经抗性药物筛选获得抗性细胞克隆和池克隆;通过RT-PCR及荧光定量PCR鉴定阳性克隆,进行平板克隆集落形成试验,以检测打靶序列沉寂LCRG1基因mRNA表达水平的效果。结果:应用PCR定点突变技术改造的pSuper载体,可被BglⅡ酶切;利用RT-PCR和荧光定量PCR检测各重组载体转染细胞池克隆LCRG1mRNA的表达发现,362组,398组,432组的基因均能封闭内源性LCRG1基因表达,尤以362组为显著;鉴定362组筛选的各个抗性克隆,发现A2和A5克隆的LCRG1mRNA表达水平明显降低。平板克隆实验结果提示362组的A2,A5和池克隆的细胞增殖能力明显强于载体和空白对照组(P<0.05)。结论:成功改建了pSuper真核表达载体;362siRNA相对其他siRNA具有较好的打靶效果,这对于应用RNAi方法研究LCRG1基因的功能和其作用分子机制具有重要的指导意义。
OBJECTIVE: To screen efficient targeting sequences of LCRG1 by RNA interference. Methods: Firstly, the pSuper vector was transformed by PCR site-directed mutagenesis. Then, five eukaryotic expression vectors targeting LCRG1 gene were constructed by using the modified vector. The constructed recombinant pSuper362, 398, 432, 789, 903 expression vector and pSuper blank vector were transfected He-la cells respectively, and the resistant cell clone and the pool clone were screened by the resistant drug screening. The positive clones were identified by RT-PCR and fluorescent quantitative PCR, The colony formation assay was used to examine the effect of silenced LCRG1 mRNA expression levels in the targeted sequence. RESULTS: The pSuper vector transformed by PCR site-directed mutagenesis was digested by BglII. The expression of LCRG1 mRNA in the transfected cells was detected by RT-PCR and real-time PCR. The results showed that the genes of 362, 398 and 432 All of which could block the expression of endogenous LCRG1 gene, especially in 362 group. Identification of 362 resistant clones showed that the expression of LCRG1 mRNA in A2 and A5 clones was significantly decreased. The result of plate clone test indicated that the cell proliferation of A2, A5 and clone in 362 group was significantly stronger than that of vector and blank control group (P <0.05). CONCLUSION: The pSuper eukaryotic expression vector was successfully reconstructed. The 362 siRNA has a better targeting effect than other siRNAs. This study is of great significance for studying the function of LCRG1 gene and its molecular mechanism by using RNAi.