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
潘晓瑜;吕延成;宋其芳;韩晋辉;朱俊访;
摘要(Abstract):
为探讨单纯疱疹病毒-2型(Herpes simplex virus type 2,HSV-2)UL27、UL54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UL27、UL54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UL27、UL54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。
关键词(KeyWords): 人疱疹病毒2型;小分子干扰RNA;UL27基因;UL54基因
基金项目(Foundation): 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)
作者(Author): 潘晓瑜;吕延成;宋其芳;韩晋辉;朱俊访;
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