人IGF-I基因慢病毒介导RNA干扰有效靶点的设计及筛选

Design and screening of clip siRNA lentiviral vector of human IGF - I

目的 设计并筛选人IGF-I有效RNA干扰片段,构建IGF-I-siRNA慢病毒表达载体.方法 对人IGF-I基因编码区分析、筛选序列4条,阴性对照序列1条.与pGCL-GFP质粒重组后,转染大肠杆菌感受态细胞.阳性克隆经过鉴定后得到正确重组子,制备编码慢病毒颗粒的重组病毒质粒及其两种辅助包装原件载体质粒,共转染293T 细胞,体外直接感染表达IGF-I的人血管平滑肌细胞.通过用Realtime-PCR检验干扰效果.结果 ①成功地合成四条编码发夹siRNA序列的单链DNA,并将其克隆到pGCSIL-GFP载体中,构建重组质粒PscSI-1、2、3、4,及无关基因的重组质粒PscNC,酶切鉴定和测序证实载体构建成功;②Realtime-PCR检测IGF-I的mRNA表达,发现重组质粒PscSI-1转染人血管平滑肌细胞后IGF-I mRNA表达最低,重组质粒PscNC转染组细胞内IGF-I mRNA的表达量与空白对照的水平接近（P〉0.05）.结论 成功构建了小的发夹式IGF-I慢病毒表达载体.在人IGF-I序列位点1010～1028 bp、序列为ACCTTGTCTAAGTGGTTTA可以在人血管平滑肌细胞中实现对人IGF-I基因表达的有效沉默.

Objective To construct the clip siRNA lentiviral vector to clarify its functions. Methods 4 gene sequences and 1 non - sence control gene sequence were consistent with the screening feature described in previous articles, and the sequences were synthesized with the help of Genechem Company. The sequences were amplificated in Bacterium coli after recombination with pGCL - GFP plasmid. After PCR and sequencing identification, the positive clones were packaged into viral host 293T cells by using liposome - mediated transfection, and we collected supematant to infect human coronary artery vascular smooth muscle cells （hCASMCs） in vitro. We detect the expression of IGF -I by using Reahime- PCR. Results PCR and DNA sequencing showed that the oligonucleotides were correct. siRNA lentiviral vector can reduce significantly the expression of IGF - I mRNA and protein by 65%, and effectively silence IGF - I gene in cultured cells. Conclusion We designed and re - combinated valid siRNA of Human IGF - I, and packaged lentiviral vector. The construction of IGF - I - siRNA lays foundation for the study of the gene function.