shRNA靶向干扰UL27、UL54基因对HSV-2复制的影响  

Effects of shRNA targeting interference of UL27 and UL54 genes on HSV-2 replication

作　　者：潘晓瑜[1] 吕延成 宋其芳[3] 韩晋辉[1] 朱俊访[1] PAN Xiaoyu;LYU Yancheng;SONG Qifang;HAN Jinhui;ZHU Junfang(Guangdong Food and Drug Vocational College,Guangzhou 512000,China;Zhuhai Campus,Zunyi Medical University,Zhuhai 519041,China;College of Life Science and Technology,Jinan University,Guangzhou 512000,China)

机构地区：[1]广东食品药品职业学院,广州512000 [2]遵义医科大学珠海校区,珠海519041 [3]暨南大学生命科学技术学院,广州512000

出　　处：《生物学杂志》2021年第1期29-32,共4页Journal of Biology

基　　金：2018年广东高校省级重点平台和重大科研项目(2018GKQNCX082);广东食品药品职业学院自然科学研究项目(2017ZR18);广东省医学科学技术研究基金项目(A2019291)。

摘　　要：为探讨单纯疱疹病毒-2型(Herpes simplex virus type 2,HSV-2)UL 27、UL 54基因的重组表达载体shRNA(small hairpin RNA,shRNA)联合干扰对HSV-2复制的影响,将重组质粒表达载体转染HEK293细胞,利用流式细胞术检测重组质粒转染HEK293的效率,实时荧光定量PCR(Quantitative Real-time PCR)和蛋白免疫印迹(Western Blot)方法检测UL 27、UL 54基因及其蛋白的表达情况。此外,采用TICD 50(50%tissue culture infective dose,TCID 50)法和噻唑蓝(methyl thiazolyl terazolium,MTT)比色法检测病毒的滴度变化和HEK293细胞的存活率。结果检测显示:UL27 shRNA75+UL54shRNA1081联合干扰组的UL 27、UL 54基因mRNA的表达抑制率分别为(88.10±1.95)%和(85.53±2.28)%,gB蛋白、ICP27蛋白表达及子代病毒滴度均明显降低,HEK293细胞的存活率为(97.30±2.91)%,与单干扰组比较差异均有统计学意义(P<0.05)。说明UL27shRNA75和UL54 shRNA1081这两个靶点联合干扰能显著抑制HSV-2在HEK293细胞中复制,为今后的HSV-2多基因的治疗提供研究基础。To investigate the effects of recombinant expression vector shRNA(small hairpin RNA)of HSV-2(Herpes simplex virus type 2)genes(including UL27 and UL54 genes)on HSV-2 replication,the recombinant expression vector shRNAs were transfected into HEK293 cells with liposome,and the transfection efficiency was detected by flow cytometry.The relative expression levels of UL27 and UL54 mRNA were detected by reverse transcription-real-time fluorescence quantitative PCR(RT-qPCR).The expression levels of UL27 and UL54 protein were detected by Western Blot,the titer of progeny virus was determined by TICD 50,and the cell survival rate was detected by MTT method.The results showed that the inhibition rates of UL27 and UL54 mRNA expression in the group of UL27 shRNA75+UL54 shRNA1081 were(88.10±1.95)%and(85.53±2.28)%,respectively.And the expression of gB protein and ICP27 protein and the titer of progeny virus were decreased significantly.The survival rate of HEK293 cells was(97.30±2.91)%.Compared with the single interference groups,the difference was statistically significant(P<0.05).These results indicated that the combined interference of UL27shRNA75 and UL54shRNA1081 could significantly inhibit the replication of HSV-2 in HEK293 cells,which will provide a basis for the future research of HSV-2 multigene therapy.

关 键 词：人疱疹病毒2型 小分子干扰RNA UL27基因 UL54基因 

分 类 号：Q78[生物学—分子生物学]