人VEGF基因慢病毒介导RNA干扰有效靶点的设计及筛选

目的:设计并筛选人血管内皮生长因子(VEGF)有效RNA干扰片段,构建VEGF慢病毒表达载体。方法:对人VEGF基因编码区分析,筛选序列3条,阴性对照序列l条,通过连接线性化的plenti6.3-MIR载体,构建miRNA慢病毒载体质粒,并转化至感受态细胞DH5α,进行测序验证。在脂质体介导下转染293T细胞,包装生产慢病毒,测定其滴度。慢病毒载体转染人肝癌细胞MHCC97L,用Real-time PCR检测干扰效果。结果:测序证实3个VEGF基因RNAi慢病毒载体质粒构建成功。慢病毒载体经293T细胞包装成功,测定病毒的滴度分别为3.23×109、3.30×109、3.73×109TU/mL。3个慢病毒载体转染人肝癌细胞MHCC97L后,VEGF基因在mRNA水平受到抑制,其中miR-200序列效果最佳,对VEGF基因表达的干扰效率可达72%。结论:成功构建并筛选了人VEGF基因RNAi慢病毒载体及有效靶点,为进一步深入研究VEGF基因与抗肿瘤药物药效关系提供实验基础。

Design and screening of RNA interference lentiviral vector of human VEGF

Objective： Design and screening of clip siRNA lentiviral vector to clarify its func-tions. Methods： Three gene sequences and one non-sence control gene sequence were consis- tent, and then were connected to linear plenti6.3 - MIR carriers in order to construct the miRNA lentiviral vector plasmid. The plasmid was transformed to DH5 a cells, and the sequence were veri- fied; 293T cells were infected by the lentiviral vector plasmid with liposome mediated, lentiviral vec- tors were packaged, and the titer was measured. The human liver cancer cells MHCC97L were in- fected by the lentiviral vectors, and the jamming effects to VEGF expression were valued by Re- al-time PCR. Results： Three VFGF RNAi lentiviral vector plasmids were constructed successfully. The lentiviral vectors were packed successfully, and the virus titers were 3.23 × 109,3.3 × 109,3.73 × 109 TU/mL Compared to the others, the miR-200 sequence had more significant efficiency and could silence 72% of VEGF gene. Conclusion： The lentiviral RNAi vector of human VEGF gene is constructed successfully, and expressed effectively in MHCC97L cells. The optimal efficient siRNA sequence was screened out, which will provide experimental basis for researching the role of VEGF gene in the effect of antitumor drugs.