双shRNA表达载体对HSV-2复制的干扰作用  

The interference effects of Duo-shRNAs expression vectors on HSV-2 replication

作　　者：向红英[1] 兰小松 吕延成[1] Xiang Hongying;Lan Xiaosong;Lyu Yancheng(Department of Biochemistry,Zhuhai Campus,Zunyi Medical University,Zhuhai Guangdong 519040,China)

机构地区：[1]遵义医学院珠海校区生物化学教研室,广东珠海519040

出　　处：《遵义医学院学报》2018年第6期690-694,共5页Journal of Zunyi Medical University

基　　金：贵州省科学技术基金资助项目(NO:黔科合LH字[2014]7581)

摘　　要：目的利用带有多个启动子的质粒p Genesil10-3p,构建针对HSV-2靶基因UL27和UL54的双短发卡RNA(Duo-shRNA)表达载体,探讨双shRNA表达载体对双靶基因的干扰作用以及对HSV-2病毒增殖的影响。方法 DuoshRNA通过脂质体转染HEK293细胞后接种HSV-2,采用四唑盐(MTT)比色法检测细胞存活率,终点滴定法检测细胞上清液中病毒滴度,QPCR(Quantitative Real-time PCR,QPCR)检测对靶基因mRNA的抑制率,蛋白质印迹(Western blot,WB)检测靶蛋白的表达量。结果结果表明Duo-shRNA对于细胞存活率、抑制HSV-2复制以及干扰靶基因表达方面都优于单-shRNA,可以达到多个单基因-shRNA联合作用的干扰效果。结论利用双启动子的方法构建了针对HSV-2的UL27和UL54的shRNA质粒表达载体,更好的抑制HSV-2病毒靶基因mRNA表达水平以及病毒在HEK293细胞中增殖,实现了多个单基因的shRNA干扰效应的相互作用。Objective Constructed a kind of Plasmid vector for expression Duo-shRNA aimed to HSV-2's UL27 and UL54,the interference effects on the target genes and effects on the proliferation of HSV-2 virus were investigated.Methods The Duo-shRNA plasmid expression vector was transfected into the HEK293 cells by liposomes and then inoculated with HSV-2.We detected the survival ratio of cells with the methyl thiazolyl tetrazolium(MTT)colorimetric method,the viral titer in supernatant of cell culture was determined by end point assay,the mRNA level of HSV-2 UL27 and UL54 were detected with quantitative real-time fluorescent PCR(QPCR).The expression of target protein was detected by Western blot(WB).Results The results showed that Duo-shRNA is superior to single-shRNA for cell viability,inhibition of HSV-2 replication and interference in gene expression,it can achieve the interference effect of multiple single-gene-shRNA interactions.Conclusion The plasmid expressing vector of UL27 and UL54 for HSV-2 was constructed by using the double promoter method,it can better inhibit HSV-2 target gene expression and virus proliferation in HEK293 cells,and realize the interaction of shRNA interference effects of multiple single genes.

关 键 词：Ⅱ型单纯疱疹病毒 RNA干扰 UL27基因 UL54基因 短发卡RNA 

分 类 号：R343[医药卫生—基础医学]