ALR siRNA慢病毒载体最佳干扰序列的筛选及构建

Screening and construction of lentiviral vector for optimal siRNA sequence targeting to ALR gene

目的筛选并构建肝再生增强因子(ALR)基因的最佳RNA干扰(RNA interference,RNAi)慢病毒表达载体,为研究ALR基因表达下调后对肝癌细胞生物学特性的影响提供依据。方法针对ALR基因序列设计并合成4个siRNA靶点,酶切连接GV118-GFP载体,PCR筛选阳性克隆,测序鉴定;在包含ALR基因的质粒中以PCR法扩增出ALR基因,克隆至慢病毒载体GV143,构建ALR基因的过表达质粒,将RNAi重组慢病毒载体和ALR的基因表达质粒共转染293T细胞,用western blot检测其对目的基因ALR的敲减效率。结果酶切鉴定证实成功构建了ALR RNAi慢病毒载体及ALR基因过表达载体。不同浓度干扰质粒(0.4μg和0.8μg)的western blot结果显示,相较于阴性对照组,KD1组ALR蛋白的表达水平(0.51±0.02,0.36±0.09)、KD2组ALR蛋白的表达水平(0.65±0.04,0.49±0.02)、KD3组ALR蛋白的表达水平(0.62±0.07,0.52±0.02)均降低(P均<0.05),而以KD1组蛋白表达量最低。结论成功筛选并构建了能有效沉默ALR基因的最佳RNAi重组慢病毒载体,为后续实验奠定基础。

Objective To screen and construct a lentiviral vector of most effective siRNA targeting to augmenter of liver regeneration （ALR） gene for providing powerful tool to investigate the effect of down-regulation of ALR gene expression on biological characters of hepatoma cells. Methods Four specific siRNA sequences targeting to ALR gene were designed and synthesized. The siRNA se- quences were cloned into GVll8-GFP lentiviral vector, and the vectors were identified by PCR and DNA sequencing. The ALR gene was amplified from the plasmid which contains ALR gene by PCR. The ALR gene fragment was inserted into lentivirus vector GV143 to construct ALR gene-expressing vector. The ALR gene-expressing vector was then transfected into 293T cells with the siRNA recombinant lentiviral vectors. Western blot was performed to determine the silence efficacy for ALR gene. Results Following digestion with re- striction enzyme, both PCR and DNA sequencing demonstrated that the siRNA-recombinant lentiviral vectors and ALR gene-expressing vector were constructed successfully. Western blot results with different concentrations of interference plasmid （0.4 μg and 0.8 μg） revealed that all the levels of ALR protein in the group KD1 （0.51 ±0.02, 0.36 ±0.09）, KD2 （0.65±0.04, 0.49±0.02） and KD3（0.62 ±0.07, 0.52 ±0.02） were lower than that in control group （P 〈0.05）. The lowest level of ALR was found in KD1 group. Conclusion The optimal siRNA recombinant lentiviral vector targeting to ALR gene was successfully screened and constructed, which provided effective tool for further study.