大鼠CD86基因RNAi慢病毒载体的构建与功能鉴定

目的 构建大鼠CD86基因的RNAi慢病毒载体并在大鼠原代培养树突状细胞(DC)上鉴定其基因沉默效率.方法 将筛选获得的大鼠CD86基因特异性siRNA靶点,合成短发卡结构shRNA序列并退火成双链DNA,与pgC-GFP慢病毒载体重组形成shRNA表达载体,利用PCR和测序鉴定获得连接正确的克隆.经由293T细胞包装shRNA慢病毒颗粒,随后将其感染原代培养的大鼠Dc细胞,采用real-time PCR和Western blot的方法检测靶基因在mRNA和蛋白水平的沉默效率.结果 构建的慢病毒载体shRNA的PcR鉴定和测序正确,shRNA慢病毒颗粒感染大鼠DC细胞后CD86基因的mRNA表达量较阴性对照载体慢病毒感染组下降了90.6%;蛋白表达显著抑制.结论 成功构建了大鼠CD86基因的shRNA慢病毒表达载体,能够在大鼠DC细胞上有效沉默靶基因.

Construction and identification of RNAi lentiviral vector targeting rat CD86 gene

Objective To construct an RNAi lentiviral vector targeting rat CD86 gene and detect its effect of gene silencing in dendritic cells. Methods The effective sequence of siRNA targeting rat CD86 gene was confirmed in our previous work. DNA oligo containing short hairpin frame was synthesized and re-annealed, and then cloned into pGCL-GFP lentivind expression vector. PCR and sequencing analysis were made for verifying the positive clones. The virus packaging plasmids were transfected into 293T cells to har-vest shRNA lentivirus. After infection in dendritic cells, real-time PCR and Western blot were performed to determine the expression level of CD86 at mRNA and protein of NC( negative control virus). Results PCR and sequencing analysis revealed that shRNA plasmid was correctly constructed. Virus with a titer of 2×108 TU/ml was successfully packaged. CD86 expression in dendritic cells can be knockdown at both mRNA and protein level by virus infection as characterized by 90.6% decrease of mRNA and significant inhibition of protein compared with NC. Conclusion The recombinant lentiviral shRNA expressing vector targeting rat CD86 gene has been successfully constructed and packaged. CD86 mRNA and protein can be effectively down-regulated in dendritic cells.