核糖体结合蛋白1干扰慢病毒载体的构建及其对人肝癌细胞的干扰效应

Construction of interference lentiviral vector targeting ribophorin 1 and its interference effect on human hepatoma cells

目的构建核糖体结合蛋白1(RPN1)的干扰慢病毒载体,建立下调RPN1表达的肝癌细胞系。方法运用分子克隆针对RPN1基因构建干扰慢病毒载体,通过与病毒包装辅助质粒共感染293T细胞包装病毒,高速离心浓缩病毒,免疫荧光方式测定病毒滴度,感染Huh7.5.1肝癌细胞系,实时荧光定量PCR(RTqPCR)测定病毒干扰效果。结果成功构建并包装RPN1RNA干扰慢病毒,病毒滴度为2×10~9 TU/mL,感染Huh7.5.1细胞后阳性率大于90.0%。相对于阴性对照,RPN1RNA干扰慢病毒组RPN1基因表达敲减效率达到92.4%(P<0.05)。结论成功构建并包装了RPN1干扰慢病毒,建立下调RPN1表达的人肝癌细胞株。

Objective To construct a interference lentivirus targeting ribophorin 1(RPN1),and establish a hepatocellular carcinoma cell line that down-regulates the expression of RPN1.Methods A interference lentivirus vector targeting the RPN1 gene was constructed by molecular cloning.The interference lentivirus was packed from 293T cells by coinfection with virus packaging auxiliary plasmids.After high speed centrifugation,the virus was concentrated and its titer was detected by immunofluorescence.The hepatoma cell line Huh7.5.1 was infected with the interference lentivirus and the interference effect of the virus was determined by RT-qPCR.Results The RPN1 RNA interference lentivirus was successfully constructed and packaged.The titer of virus was 2×10^9 TU/mL,and the positive rate after infection of Huh7.5.1 cells was more than 90.0%.Compared with negative control,the knockdown rate of RPN1 gene expression in RPN1 RNA interference lentivirus group was 92.4%(P<0.05).Conclusion RPN1 interfering lentivirus was successfully constructed and packaged,and a human hepatocellular carcinoma cell line with down-regulation of RPN1 expression was established.