靶向PCSK9基因shRNA慢病毒载体的构建及其对PCSK9基因表达的沉默

目的设计以PCSK9基因为靶点的短发夹状RNA（shRNA），构建重组慢病毒载体并鉴定此RNA干扰体系对PCSK9基因表达的影响。方法将3条人PCSK9基因的shRNA片段插入至慢病毒载体pLentilox3．7，与pCDNA3-hPCSK9．FLAG质粒用Lipofectamine2000共转染293细胞，Western blotting法鉴定出最有效的shRNA。此重组质粒与pCre—VSV-G、pLoxp．CMV—R8．91经293细胞包装后，产生的重组慢病毒感染293细胞，48h后转染pCDNA3．hPCSK9-FLAG质粒，Western blotting法检测人PCSK9基因表达的情况。结果经双酶切鉴定，构建了PCSK9shRNA慢病毒载体pLentilox—hPC—SK9，鉴定出hshRNA-2为最有效的shRNA。重组的慢病毒能明显的抑制人PCSK9的表达。结论慢病毒介导的shRNA干扰技术可特异性的阻断PCSK9的表达，为进一步探讨PCSK9特异性的shRNA治疗脂代谢异常疾病奠定了基础。

Construction of small hair RNA lentiviral vectors targeting PCSK9 gene and its silent effects on PCSK9 gene

Objective To construct lentiviral vector-based short hairpin RNA （shRNA）targeted at human PCSK9 and evaluate its inhibitory effects on the expression of PCSK9 gene. Methods shRNAs targeted at human PCSK9 RNA were designed and cloned into plentilox 3.7 plasmid. The three recombinant plasmids（ shRNA-1, shRNA-2 and shRNA-3）were identified by enzyme digestion and sequencing. Positive plasmids were co-transfected with pCDNA3-PCSK9-FLAG to 293 cells to test their knockdown functions using Western blotting methods. Cells transfected with empty plentilox3.7 was used as the control. Recombinant lentivirus was produced by co-transfeeting hshRNA-2,pCre-VSV-G and pLoxp-CMV-RS. 91 into 293 packaging cells. 293 cells were then infected with lentiviral hshRNA-2, lentiviral control shRNA, or empty lenti- virus. After 48 hours, the cells were transfected with pCDNA3-hPCSK9-FLAG and the PCSK9 expression was measured by Western blotting methods. Results The shRNA lentiviral vectors targeting PCSK9 gene were confirmed by double enzyme digestion and sequencing. HshRNA-2 showed high inhibitory efficacy. Lentiviral hshRNA-2 decreases the expression of human PCSK9 protein markedly. Conclusions lentiviral vector-based shRNA targeted at human PCSK9 could knock down their gene expressions specifically by RNA interference （RNAi） technology, which provided a useful tool for investigating PCSK9-specific shRNA in regulating lipid metabolism.