VEGF干扰慢病毒载体的构建及其在MHCC97L细胞中的表达

Construction of VEGF gene lentiviral RNA interference vector and its expression in MHCC97L cells

目的构建VEGF基因RNA干扰(RNAi)慢病毒载体并实现其在人肝癌细胞MHCC97L中表达。方法根据人VEGF基因序列,设计并合成包含靶序列的互补DNA链,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RNAi组(MHCC97-200)、阴性对照组(MHCC97-NEGA)和空白对照组(MHCC97-空白)中VEGF的表达情况,确定其干扰VEGF表达的有效性。结果测序证实慢VEGF基因RNAi病毒载体质粒构建成功。慢病毒载体经293T细胞包装成功,测定病毒的滴度为3.23×109TU/mL。慢病毒载体转染人肝癌细胞MHCC97L 48 h后,VEGF基因在mRNA水平抑制了72.2%。结论 VEGF基因RNAi慢病毒载体构建成功,并实现在人肝癌细胞MHCC97L中的表达。

Objective To construct the lentiviral RNA interference（RNAi） vector of human VEGF gene and realize the expression in human liver cancer cells MHCC97L.Methods According to the sequence of human VEGF gene,the complementary DNA chains which contain the target sequence were designed and synthesized,and then were connected to linear plenti6.3-MIR carriers in order to construct the miRNA lentiviral vector plasmid.The plasmid was transformed to DH5α cells,and the sequence were verified;293T cells were infected by the lentiviral vector plasmid with liposome mediated,lentiviral vectors were packaged,and the titer was measured.The human liver cancer cells MHCC97L were infected by the lentiviral vectors,and the VEGF expression in RNAi group（MHCC97-200）,the negative control group（MHCC97-NEGA） and the blank control group（MHCC97-blank） were detected by Real-time PCR,to ensure the jamming effect of lentiviral vector plasmid to VEGF expression.Results The VEGF RNAi lentiviral vector plasmid was constructed successfully.The lentiviral vector was packed successfully,and the virus titer was 3.23×109 TU/mL.The VEGF gene expression was reduced by 72.2% at mRNA level 48 h after the human liver cancer cells MHCC97L were infected by the lentiviral vectors.Conclusion The lentiviral RNAi vector of human VEGF gene is constructed successfully,and expressed effectively in MHCC97L cells.