特异性针对小鼠VEGFa基因的siRNA慢病毒载体的构建

目的 以小鼠VEGFa基因为靶基因，构建携带短发夹状干扰RNA(siRNA)的慢病毒载体。方法 设计并合成三对包含靶序列和一对针对无关序列的互补寡核苷酸链，克隆到pRNAT-U6.2/Lenti质粒；从小鼠NIH/3T3细胞扩增、纯化VEGFa cDNA，构建pCDNA-VEGF质粒；实验分6组，pCDNA-VEGF质粒或/和siRNA慢病毒质粒共转染NIH/3T3细胞，筛选最有效siRNA慢病毒表达质粒；将筛选siRNA慢病毒表达质粒和pRNAT-Negative质粒与慢病毒包装质粒混合物混合，共转染293T细胞，48h后收集上清。结果成功构建针对小鼠VEGFa基因的siRNA慢病毒质粒和质粒pCDNA-VEGF；慢病毒质粒有效抑制pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa表达水平，pRNAT-siRNA3抑制作用最明显，VEGFa mRNA和蛋白表达分别下降80.0%和66.3%；纯化慢病毒滴度均为1×108ifu/mL。结论 成功设计、筛选、生产针对小鼠VEGFa基因沉默的特异性siRNA慢病毒。

A recombinant lentiviral vector carrying siRNA targeting the endothelial vascular growth factor a gene in mice

To construct a recombinant lentiviral expression vector carrying small interference RNA (siRNA) with the mouse endothelial vascular growth factor a (VEGFa) gene serving as the target. Methods  ① Three target sequences of mouse VEGFa gene and an irrelevant sequence were designed. Four couples of complementary oligonucleotides with hairpin loop of siRNA were synthesized. After annealing, the DNA fragments were ligated into the plasmid pRNAT U6.2/Lenti. ② VEGFa cDNA was cloned from NIH/3T3 strain and subcloned into the plasmid pKCDNA-EF1-Puro. ③ The pCDNA-VEGF plasmid and siRNA lentiviral plasmids were co-transfected into the NIH/3T3 cells and assigned into six groups, the effects of RNAi on VEGFa expression were determined 48h later; ④ The lentiviruses were produced by 293T cells following cotransfection of the pRNAT-siRNA3 plasmid or the pRNAT-negative plasmid with three package plasmid compounds. After 48h, the supernatant was collected and the recombinant lentiviruses titers were determined. Results ① Three lentiviral plasmids of siRNA specific to the VEGFa gene and one negative plasmid were constructed. ② The plasmid pCDNA-VEGF was constructed. ③ In the pRNAT-siRNA3 plasmid cotransfection group, expression of VEGFa mRNA and protein was decreased by approximately 80.0% and 66.3%, respectively.④ The lentivirus titer reached 1×108ifu/ml. Conclusion  The recombinant lentiviruses,which express siRNA hairpin aiming at mouse VEGFa gene, have been constructed.